Methods and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom

ABSTRACT

Blood-derived plastic articles prepared from compositions including blood and, in some embodiments, at least one crosslinking agent and/or at least one biological response modifier, that can be useful for biological applications such as wound repair and tissue grafts; methods of making and using the same; methods for assessing the concentration of a biological response modifier in an article; and systems for preparing blood-derived plastic articles are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/104,728, filed Apr. 17, 2008, which is a continuation-in-part of U.S.patent application Ser. No. 11/873,751, filed Oct. 17, 2007, whichclaims the benefit of U.S. Provisional Patent Application Ser. Nos.60/852,368, filed Oct. 17, 2006, and 60/961,580, filed Jul. 23, 2007,all of which are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The invention pertains to bioplastics for patient implantation orapplication, made at least in part from patient tissue or fluids such asplasma.

BACKGROUND OF THE INVENTION

Fibrin-based plastics were invented in the 1940s as part of a U.S.Defense-sponsored research program to develop medical strategies forwounded military personnel. For example, fibrin-based plastics weredeveloped out of the human blood program led by Edwin Cohn at HarvardUniversity. John Ferry, then at Woods Hole, led the group that wasinvolved in developing fibrin elastomers. As a result of this work,elastomeric sheet forms of fibrin were developed and used successfullyin neurosurgical applications, burn treatments, and peripheral nerveregeneration. See, for example, Ferry, J. D. et al., Clin. Invest.23:566-572 (1944); Bailey, O. T. et al, J. Clin. Invest. 23:597-600(1944); Cronkite et al., JAMA, 124:976-8 (1944); and Ferry J. D. et al.,Am. Chem. Soc J. 69:400-409 (1947). Hard fibrin plastics were fabricatedinto implants and were finding clinical success as early as the 1940s.See, for example, U.S. Pat. Nos. 1,786,488, 2,385,802, 2,385,803,2,492,458, 2,533,004, 2,576,006, 3,523,807, 4,548,736 and 6,074,663, allincorporated herein by reference. Research sponsored by the Hungariangovernment led to the development of similar products in the 1950sthrough the early 1970s. Forms of hard plastic fibrin were demonstratedto have clinical efficacy in orthopedic applications of boneresurfacing. See, for example, Zinner, N. et al., Acta Med. Acad. Sci.Hung. 7:217-222 (1955); Gerendas, M., Ther. Hung., 7:8-16 (1959); andGerendas, M., Chapter 13 in Fibrinogen, Laki, K., Ed., Marcel Dekker,New York, pp. 277-316 (1968).

Despite the efficacy of fibrin products, concerns about diseasetransmission from purified human fibrinogen from pooled plasma remained.However, during the late 1970s and thereafter, fibrin was developed as atissue glue and sealant, and although this application required purifiedhuman fibrinogen, new techniques had been developed to ensure the safetyof blood products. Consequently, fibrin-based glues and sealants havebeen used in clinical practice for over twenty years in Europe (andsince 1998 in the United States) with no disease transmission concerns.Recently, the development of recombinant human fibrinogen and thrombinand purified salmon fibrinogen and thrombin have helped further toaddress concerns over both safety and market availability. See, forexample, Butler S. P. et al., Transgenic Res. 13:437-450 (2004);Prunkard D. et al., Nat. Biotechnol. 4:867-871 (1996); Butler S. P. etal, “Thromb Haemost. 78:537-542 (1997); U.S. Pat. No. 5,527,692; U.S.Pat. No. 5,502,034; U.S. Pat. No. 5,476,777; U.S. Pat. No. 6,037,457;U.S. Pat. No. 6,083,902; and U.S. Pat. No. 6,740,736. Autologoussealants and glues are also available (see for example U.S. Pat. No.6,979,307).

Despite such advances in the field, interest in the use of proteinbioplastics in plastic forms, such as fibrin elastomers, hassignificantly declined over time. Silicone rubber sheets, which wereintroduced in the 1960s and 1970s, have supplanted fibrin elastomericsheets in the clinic, despite inherent problems with their permanence.There are also limitations with current synthetic bioresorbableplastics, such as polyurethane, polylactic acid (PLA),polylactic-co-glycolic acid (PLGA), polyglycolic acid (PGA) andpolycaprolactone. These polymers degrade in the body by hydrolysis, viabulk degradation, or through surface erosion, all of which operateindependently of the surrounding biological environment. The inabilityof these polymers to degrade in response to cellular invasion and topromote directly the in-growth of host tissues remains a profoundlimitation of these types of bioresorbable implants.

In contrast, protein bioplastics can degrade in response to cellularproteolytic processes so that degradation occurs in concert with thegrowth and healing of host tissues.

Also, many synthetic materials do not inherently bind growth factors ofinterest for therapeutic delivery options, whereas fibrin binds to manygrowth factors directly and indirectly through molecular interactionswith growth factors, including those with heparin binding domains.However, fibrin materials—including certain of the present inventors'own fibrin-based plastics based on purified fibrinogen/thrombin frompooled human or animal plasma—have certain constraints or limitations,such as not inherently containing endogenous growth factors. Moreover,fibrin materials of the prior art are very expensive, especially whenprepared from human sources and with the required large amounts ofstarting material necessary to give desired yields. Commonly usedsynthetic materials, such as bioresorbable polymers, can be associatedwith undue inflammatory interactions, whereas these interactions wouldbe less pronounced if one were to use protein-based plastics.Protein-based plastics, such as those based upon purified fish- orbovine-derived fibrinogen, are potentially less expensive—althoughsimilar to its purified human counterpart in not containing human growthfactors—yet disease transmission and immuno-sensitization with repeateduse are potential major drawbacks due to its xenogenic source.Analogously to plastic implants, allogeneic bone grafts also haveseveral limitations, including high variability of graft quality fromdonor to donor. This variability arises from several factors, includingamount of active endogenous growth factors in each donated graft, andthere are no practical means for quality assessment (QA) and/or qualitycontrol (QC) of allogeneic bone graft materials with respect to thesegrowth factors.

To date, methods and compositions previously developed for bioplastics,including but not limited to fibrin, elastin, etc., are not sufficientlyadaptable for modern clinical use. Prior efforts to chemically crosslinkfibrin-based bioplastics were either labor-intensive post-fabricationmethods, which generally created unwanted effects such as swelling,and/or used toxic crosslinking agents such as formaldehyde.Manufacturing methods developed for certain protein-based bioplasticsrequired high temperatures (i.e., as high as 155° C.). Such hightemperature processing can preclude the use of exogenously added drugsand proteins, as well as destroy any inherent biological activity. Inaddition, methods for making such materials porous have not beenreported or developed previously. Furthermore, steam sterilization cancompletely denature any biological activity in purified blood proteins.The problem of manufacturing bioplastics while avoiding thedisadvantages of known processing techniques, such as high temperaturesand pressures and/or difficulty in retaining desirable physicalcharacteristics of the plastics, has not been adequately addressed.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides blood-derivedplastic articles comprising at least one biological response modifier.

In some embodiments, the present invention provides blood-derivedplastic articles prepared from a composition comprising (1) blood and(2) at least one crosslinking agent selected from the group consistingof iridoid derivatives, diimidates, diones, carbodiimides, acrylamides,sugars, proteins, dimethylsuberimidates, aldehydes, Factor XIII, dihomobifunctional NHS esters, carbonyldiimide, glyoxyls, proanthocyanidin,reuterin, and mixtures thereof.

In some embodiments, the present invention provides blood-derivedplastic bone tissue articles having a Young's Modulus ranging from about0.03 GPa to about 50 GPa, measured according to ASTM Method No. D-638-03and a compressive strength ranging from about 1 MPa to about 250 MPaaccording to ASTM Method No. D-695-02a, the Young's Modulus andcompressive strength being determined at a temperature of about 25° C.and a pressure of about 101 KPa (about 1 atm).

In some embodiments, the present invention provides blood-derivedplastic tendon tissue articles having a Young's Modulus ranging fromabout 0.5 GPa to about 1.5 GPa, measured according to ASTM Method No.D-638-03, a percent strain at failure ranging from about 8% to about 16%according to ASTM Method No. D-638-03, and a stiffness-ranging fromabout 100 N/mm to about 5000 N/mm according to ASTM Method No. D-638-03,the Young's Modulus, percent strain at failure and stiffness beingdetermined at a temperature of about 25° C. and a pressure of about 101KPa (about 1 atm).

In some embodiments, the present invention provides blood-derivedplastic ligament tissue articles having a Young's Modulus ranging fromabout 100 MPa to about 1000 MPa, measured according to ASTM Method No.D-638-03 and a stiffness ranging from about 50 N/mm to about 1000 N/mmaccording to ASTM Method No. D-638-03, the Young's Modulus and stiffnessbeing determined at a temperature of about 25° C. and a pressure ofabout 101 KPa (about 1 atm).

In some embodiments, the present invention provides blood-derivedplastic cartilage tissue articles having a Young's Modulus ranging fromabout 1 MPa to about 250 MPa measured according to ASTM Method No.D-638-03, a percent strain at failure ranging from about 0.1% to about1% according to ASTM Method No. D-638-03, and a stiffness ranging fromabout 5 N/mm to about 4000 N/mm according to ASTM Method No. D-638-03,the Young's Modulus, percent strain at failure and stiffness beingdetermined at a temperature of about 25° C. and a pressure of about 101KPa (about 1 atm).

In some embodiments, the present invention provides blood-derivedplastic skin tissue articles comprising at least one biological responsemodifier, wherein the article has a Young's Modulus ranging from about0.1 MPa to about 20 MPa, measured according to the “Skin Young's ModulusTest” described below, and an elasticity ranging from about 50% to about100% according to the “Elasticity Test” described below, the Young'sModulus and elasticity being determined at a temperature of about 25° C.and a pressure of about 101 KPa (about 1 atm).

In some embodiments, the present invention provides blood-derivedplastic skin tissue articles prepared from components comprising (1)blood and (2) at least one crosslinking agent selected from the groupconsisting of iridoid derivatives, diimidates, diones, carbodiimides,acrylamides, sugars, proteins, dimethylsuberimidates, aldehydes, FactorXIII, dihomo bifunctional NHS esters, carbonyldiimide, glyoxyls, anddimethylsuberimide and mixtures thereof, wherein the article has aYoung's Modulus ranging from about 0.1 MPa to about 20 MPa, measuredaccording to the “Skin Young's Modulus Test” described below, and anelasticity ranging from about 50% to about 100% according to theElasticity Test described below, the Young's Modulus and elasticitybeing determined at a temperature of about 25° C. and a pressure ofabout 101 KPa (about 1 atm).

Methods of preparing and using the above articles also are provided.

In some embodiments, the present invention provides methods forassessing the concentration of a biological response modifier in anarticle comprising: (a) providing a range of acceptable concentrationsof a pre-determined biological response modifier for a batch of blood tobe used to prepare an article; (b) determining the concentration of apredetermined biological response modifier in a blood batch to be usedto prepare an article; and (c) comparing the concentration determined in(b) to the range of acceptable concentrations obtained from (a) todetermine if the concentration determined in (b) is above or below therange of acceptable concentrations determined in step (a).

In some embodiments, the present invention provides methods for making ablood-derived plastic article comprising: (a) collecting a quantity ofblood; (b) clotting said blood; (c) drying said clotted blood; and (d)contacting a quantity of the clotted dried blood with at least oneplasticizer to make a bioplastic dough, and shaping and heating saidbioplastic dough to make a blood-derived plastic article.

In some embodiments, the present invention provides a system forpreparing blood-derived plastic articles, comprising: a dryer for atleast partially drying blood; a powderizing device, such as a powdermiller, for milling the at least partially dried blood received from thedrying apparatus to form a blood powder; a mixer for mixing the bloodpowder with at least one plasticizer to form a molding composition; anda compression molding apparatus comprising at least one mold forreceiving the molding composition from the mixer and a vacuum degasserfor removing gas from the molding composition during molding.

In some embodiments, the present invention provides methods forpromoting healing of a skin wound comprising: applying to the skin woundsurface an effective amount of a blood-derived plastic article, whereinthe blood-derived plastic article comprises at least one biologicalresponse modifier.

In some embodiments, the present invention provides methods forpromoting healing of a tissue wound or defect comprising: applying tothe tissue wound or defect an effective amount of a blood-derivedplastic article, wherein the blood-derived plastic article comprises atleast one biological response modifier.

In some embodiments, the present invention provides methods forproviding a resorbable graft to a graft position in a subject,comprising: inserting a blood-derived plastic article into a graftposition in a subject, wherein the blood-derived plastic articlecomprises at least one biological response modifier.

In some embodiments, the present invention provides methods fordelivering stem cells to a tissue of a subject, comprising: contacting ablood-derived plastic article comprising stem cells with a tissue of asubject.

In some embodiments, the present invention provides methods forconnecting a first portion of a tissue with a second portion of atissue, comprising: contacting at least one blood-derived plasticarticle selected from the group consisting of a suture, staple and barbwith a first portion of a tissue with a second portion of a tissue, suchthat the first portion of the tissue and the second portion of thetissue are connected.

In some embodiments, the present invention provides methods for forminga blood-derived plastic film, comprising: (a) drying a blood-derivedcomposition under vacuum to reduce the water content thereof and form anat least partially dried composition; and (b) shaping the at leastpartially dried composition into a film.

In some embodiments, the present invention provides methods for forminga blood-derived plastic article, comprising: (a) lyophilizing ablood-derived composition to reduce the water content thereof and forman at least partially dried composition; (b) mixing the at leastpartially dried composition with at least one plasticizer to form amixture; and (c) shaping the mixture into a blood-derived plasticarticle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts electron micrographs taken after osteoblastic precursorcells were cultured on plasma-based plastics (PBPs) and then monitoredfor subsequent cell interactions using scanning electron microscopy;

FIG. 2 depicts samples of the present blood plasma-derived plasticarticle in which (top row) Genipin was added as a powder to thecomponents without prior alcohol solubilization of Genipin and (secondrow) Genipin solubilized in alcohol was added to the components;

FIG. 3 depicts micrographs that illustrate how smaller particle sizesenable more and better uniformity in mold fill and molded product; and

FIG. 4 is a schematic flow diagram of one embodiment of the presentmethod for making a blood plasma-derived plastic article from bloodplasma.

DETAILED DESCRIPTION

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients, thermal conditions, and soforth, used in the specification and claims are to be understood asbeing modified in all instances by the term “about”. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are approximations that mayvary depending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Furthermore, when numerical ranges ofvarying scope are set forth herein, it is contemplated that anycombination of these values, inclusive of the recited values, may beused.

Also, it should be understood that any numerical range recited herein isintended to include all sub-ranges subsumed therein. For example, arange of “1 to 10” is intended to include all sub-ranges between andincluding the recited minimum value of 1 and the recited maximum valueof 10, that is, having a minimum value equal to or greater than 1 and amaximum value of equal to or less than 10.

As used herein, the phrase “reaction product of” means chemical reactionproduct(s) of the recited components, and can include partial reactionproducts as well as fully reacted products.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombinations of the specified ingredients in the specified amounts.

As used herein, “formed from” or “prepared from” denotes open, e.g.,“comprising,” claim language. As such, it is intended that an article“formed from” or “prepared from” a list of recited components be anarticle comprising at least the recited components or the reactionproduct of at least the recited components, and can further compriseother non-recited components used during the article's formation orpreparation.

As used herein, the term “polymer” means a substance, typically of largemolecular mass, comprising structural units or monomers. Polymers can bebiological or natural materials, such as proteins, DNA, RNA, starches,fibrin and collagen, or synthetic materials, and are meant to encompassoligomers, homopolymers and copolymers. The term “prepolymer” means acompound, monomer or oligomer used to prepare a polymer, and includeswithout limitation both homopolymer and copolymer oligomers. The term“oligomer” means a polymer consisting of only a few monomer units up toabout ten monomer units, for example a dimer, trimer or tetramer.

As used herein, the term “elastomer” refers to a polymeric materialwhich at room temperature is capable of repeatedly recovering in sizeand shape after removal of a deforming force. In some embodiments, anelastomer is a material which can be repeatedly stretched to twice itsoriginal length and will repeatedly return to its approximate length onrelease of the stress.

As used herein, “plastic” refers to any substance, such as organic,synthetic, and/or processed materials that comprise polymers and can bemade into structures such as 3-dimensional constructs and 2-dimensionalconstructs, such as, for example, films, sheets, laminates, filaments,and similar structures. See, for example, U.S. Pat. No. 6,143,293. Asused herein, “hard plastic” refers to a plastic that tends to break inresponse to sufficient deformation and, thus, has small plastic and/orelastic deformation range; whereas, the term “soft plastic” refers to aplastic that readily deforms under stress without breaking, and, thus,has a large plastic and/or elastic deformation range.

As discussed above, there is a long-felt need for articles forbiological applications, such as tissue grafts or tissue repair, having,inter alia, good biocompatibility, biodegradability, ease of manufactureand/or low cost. Also, it would be desirable to provide such articleshaving biologically active response modifiers, such as hormones, growthfactors, and extracellular matrix molecules, and/or capability for drugdelivery. Articles having the ability to respond to the local cellularmilieu also are needed, with or without spatial patterns in the overallconstruct or sheet to provide such responses where desired. Also,articles are needed having desired physical properties, such as density,porosity, resorbability, and/or mechanical properties which arecompatible with the biological environment into which such an article isto be placed. More reliable, cost-effective substitute tissue and graftmaterials have remained an illusive yet important clinical goal.

In some embodiments, the present invention provides blood-derivedplastic articles which can be useful for biological applications, suchas wound or tissue repair; tissue grafts such as bone grafts, tendongrafts, ligament grafts, or skin grafts; nerve guides;prosthetics/tissue interfaces; corneal grafts; plates; screws; fixtures;guides; sutures; clips; staples; barbs; resurfacing materials; tendonrepair; and scaffolds for tissue engineering, for example for celldelivery such as stem cell delivery, to name a few. As used herein, theterm “tissue” refers to an aggregation of similarly specialized cellsunited in the performance of a particular function. Tissue is intendedto encompass all types of biological tissue including both hard and softtissue, including connective tissue (e.g., hard forms, such as osseoustissue or bone) as well as other muscular or skeletal tissue.

The articles of the present invention can possess one or more of thefollowing desirable characteristics: biocompatibility of the materialswith the host or subject; the ability of the materials to degrade inrelation to tissue regeneration; the binding of growth factors to thematerials disclosed herein, which thereby helps minimize the dosagesneeded to produce therapeutic results; the ability to easily engineerthe mechanical properties (e.g., ranging from elastic to rubbery tohard) of the materials; the ability to easily store the materials foroff-the-shelf usage; the ability to easily shape the materials at a timeand a place where the materials will be fabricated and/or clinicallyapplied (e.g., a blood bank, an operating room, a battlefield, etc.);the ability of the article to resisting tissue prolapse at theimplantation site; the ability to modulate the physiological response tothe implanted materials by incorporating other materials into the basematerial; and the ability to select blood donors of predetermined age orother desired characteristic to provide articles having predeterminedbiological characteristics.

Allogeneic transplant materials, such as bone grafts, have severallimitations, including high variability of graft quality from donor todonor. This variability arises from several factors, including amount ofactive endogenous growth factors in each donated graft, and there is nosimple means for quality assessment and/or quality control of allogeneicbone grafts with respect to such growth factors. In contrast, theblood-derived plastic articles of the present invention can be preparedfrom pooled and processed allogeneic blood plasma from many donors whichcan homogenize the material constituents of the plasma, and the amountand/or types of growth factor(s) as well as other biological componentscontained therein, can be readily assessed for quality analysis and/orquality control as discussed below.

In some embodiments, blood-derived plastic articles of the presentinvention are prepared from a composition comprising whole blood andother components, as discussed in detail below. As used herein,“blood-derived” means prepared from whole blood or blood components,such as plasma or serum.

In some embodiments, plastic articles of the present invention areprepared from a composition comprising blood plasma and othercomponents, as discussed in detail below. As used herein, “bloodplasma-derived” means prepared from blood plasma.

“Whole blood” is a body fluid (technically a tissue) that is composed ofblood cellular components suspended in a liquid called blood plasma.Blood cellular components include red blood cells (also called RBCs orerythrocytes), white blood cells (including both leukocytes andlymphocytes) and platelets (also called thrombocytes). As used herein,“blood” generally means whole blood or any fraction thereof, such asplasma or serum.

“Plasma” is defined as the fluid portion of human blood that has beencollected, stabilized against clotting and separated from the red bloodcells. Plasma can be obtained by separating plasma from blood collectedfrom blood donors or by plasmapheresis. Plasma may be obtained fromwhole blood. Blood plasma is essentially an aqueous solution containingabout 92% water, about 8% blood plasma proteins (such as serum albumin,blood clotting factors, immunoglobulins (antibodies)), various otherproteins, electrolytes, such as sodium and chloride, hormones, xymogens,proteases, protease inhibitors and trace amounts of other materials.“Serum” is defined as blood plasma from which the clotting proteins havebeen removed, i.e., without fibrinogen and other clotting factors. Alarge percentage of the proteins remaining are albumin andimmunoglobulins.

In some embodiments, the blood plasma can be “platelet-rich” or “PRP”,i.e., pre-treated prior to mixing with other components of thecomposition to increase the concentration of platelets compared to theconcentration of platelets of the blood plasma at baseline prior to suchtreatment. Platelet-rich plasma can be prepared by centrifugation andmay have at least 250,000 platelets per microliter. In some embodiments,the blood plasma can be platelet-poor. “Platelet-poor” plasma is aportion of the plasma fraction of blood having a platelet concentrationbelow baseline. Fresh frozen plasma (“FFP”) is technically the same asplatelet-poor plasma.

One embodiment of the present invention is a method of manufacturingautologous bioplastics by processing a patient's own donated bloodplasma—and products produced thereby. A typical method of making such anautologous PBP (plasma-based plastic) is as follows. Blood is collectedprior to surgery. The blood is spun down to obtain platelet-rich plasma(PRP) and/or platelet poor plasma (PPP) and/or serum, or comparablemethods such as whole blood collection or via apheresis are used tocollect plasma from the patient without having to collect whole blood.The plasma is then clotted with calcium, thrombin or other knownclotting agents, and the clotting when performed on platelet-rich plasmaforms a platelet-rich plasma gel. To make rubbery-to-hard plastics, theplatelet-rich plasma gel is first processed into a powder by drying it(this can include first removing any retained serum or not, although itis also possible to use only serum by drying it into a powder) and thenball milling or grinding or other powdering techniques. The drying stepmay or may not include lyophilization, but plasma dried “through the gelphase” for use in elastomers generally should not be lyophilized ifpossible (see below). Alternatively, a serum-free powder can be formedby first removing serum from the gel by spinning and then drying andcomminuting the remaining plasma. In general, then, the presentinvention can use whole plasma or plasma from which one or moreconstituents has been removed as desired (even to the point of onlyserum's remaining).

The source of blood (whole blood or its components such as blood plasmaor serum) used to prepare the articles of the present invention caninclude humans and other mammalian species, for example, primates,rodents and livestock such as sheep, goat, pig, horse, dog and cattle.The blood can be from autologous sources or allogeneic sources. As usedherein, “allogeneic” means that the blood is taken from differentindividuals within the same species.

In some embodiments, the source of blood can be donors between about 18and about 65 years of age, or between about 18 and about 30 years ofage, or between about 18 and about 25 years of age, or about 30 years ofage or less. In some embodiments, the source of blood can be eitherfemale donors or male donors, or both.

The blood plasma used in the present invention can be obtained fromblood using conventional methods such as centrifugation, sedimentationand filtration. Centrifugation can be carried out under any conditionswell known to those skilled in the art as suitable to sediment bloodcells (such as red and white blood cells) and cell fragments (such asplatelets), for example, at about 2800 μm for about 10 minutes. Thesupernatant plasma can be easily separated from the centrifuged cells byconventional techniques, for example by passing the supernatant plasmathrough a suitable filter, such as a microporous membrane.

The blood can be fresh liquid blood, or solid or powdered blood. In someembodiments, the blood can be at least partially dried or essentiallyfully dried, if desired. In some embodiments, the dried blood has awater content of less than about 30 weight percent on a basis of totalweight of the dried blood, or less than about 25 weight percent, or lessthan about 20 weight percent, or less than about 15 weight percent,prior to combination with the other components used to form the article(prior to mixing the composition or dough). In some embodiments, thedried blood has a water content of about 1 to about 25 weight percent ona basis of total weight of the dried blood, or about 5 to about 15weight percent, or about 8 to about 12 weight percent, or about 8 toabout 10 weight percent, prior to combination with the other componentsused to prepare the article.

The water content can be determined by various methods, for example asfollows: pre-weigh three 1.5 mL microfuge tubes that have two smallholes placed in the lids with a needle. Add approximately 100 mg ofdried blood or plasma powder to the tube and record the mass. Place thetubes in an 80° C. oven for 48 hours. Remove the tubes, allow to cool toroom temperature (about 25° C.), and weigh the tubes. Subtract the driedblood or plasma powder mass from the original powder mass, divide thatvalue by the original powder mass, and multiply by 100%. The average ofthe percents obtained from the three microfuge tubes is the average %mass of water in the sample, i.e., water content.

The composition or dough is the combination of blood, plasticizer (ifpresent) and any other components that are mixed prior to plastificationprocessing. In some embodiments, the blood can comprise water which canfunction as a plasticizer. Alternatively or additionally, in someembodiments the composition can comprise one or more plasticizers suchas are discussed in detail below.

In some embodiments, the blood is dried through the gel phase, byremoving a portion of the water that is inherent from the originalplasma clot which can represent about 0.1 to greater than about 25% byweight of the starting material. As used herein, the phrase “driedthrough the gel phase” means that the dried blood has a water content ofabout 0.01 to about 25 weight percent, or about 0.01 to about 10 weightpercent, or about 0.01 to about 5 weight percent based upon total weightof the dried blood.

In some embodiments, the average particle size of the at least partiallydried blood is less than about 1000 microns (μm) prior to mixing withother components of the composition, or less than about 500 μm, or lessthan about 150 μm, or less than about 38 μm, or about 1 μm to about 500μm, or about 38 μm to about 500 μm, or about 38 μm to about 150 μm.

In some embodiments in which the average particle size of the particlesis greater than one micron, the average particle size can be measured bymesh sieving or according to known laser scattering techniques. Forexample, the average particle size of such particles is measured using aHoriba Model LA 900 laser diffraction particle size instrument, whichuses a helium-neon laser with a wavelength of 633 nm to measure the sizeof the particles and assumes that the particle has a spherical shape,i.e., the “particle size” refers to the smallest sphere that willcompletely enclose the particle.

In some embodiments in which the size of the particles is less than orequal to one micron, the average particle size can be determined byvisually examining an electron micrograph of a transmission electronmicroscopy (“TEM”) image, measuring the diameter of the particles in theimage, and calculating the average particle size based on themagnification of the TEM image. One of ordinary skill in the art willunderstand how to prepare such a TEM image. The diameter of the particlerefers to the smallest diameter sphere that will completely enclose theparticle.

The blood can be treated prior to incorporation into the composition,for example by fresh-frozen preparation, cryoprecipitated preparation,lyophilized preparation or concentrated preparation. Fresh-frozen plasmacan be obtained by centrifuging the blood at about 2,000 rpm for about10 minutes to separate out blood cells and cell fragments and freezingthe remaining liquid portion at the temperature of from about −18° C. orlower, or about −18° C. to about −40° C. The centrifugation can becarried out within six hours of blood collection. For use, thefresh-frozen plasma can be thawed out in a warm water bath at atemperature of about 30° C. to 37° C. Cryoprecipitated plasma can beobtained by thawing fresh-frozen plasma at a temperature of 4° C. toform white precipitate (cold precipitated protein), isolating the formedprecipitate and refreezing it at a temperature of about −18° C. to −40°C. For use, the cryoprecipitated preparation can be thawed out byrefrigerating at a temperature of from 1° C. to 6° C. overnight.

In some embodiments, the blood can be obtained from commercial sources,such as blood banks. These preparations are derived from units of humanblood or blood plasma which have been tested to elicit noantigen-antibody reaction, for example, non-reactive for antibodies tohepatitis B surface antigen (HBsAg) and hepatitis C (HCV) antibody andnegative for antibodies HIV-1 and HIV-2 viruses. All units of bloodplasma or serum used to prepare such preparations are certified free ofpathogens.

To reduce the potential risk of transmission of infectious agents, thepreparation may be treated with an organic solvent/detergent mixture,such as tri(n-butyl)/phosphate/polysorbate 80 designed to inactivateenveloped viruses such as HIV, hepatitis B and HCV. The inactivation andremoval of viruses can be enhanced by nanofiltration. In someembodiments, the plasma can be prepared by pasteurization of a liquidplasma fraction. Alternatively, the whole blood can be purified and theresultant plasma can be powdered by heating, lyophilization or othersuitable drying techniques.

In some embodiments, the plasma is at least partially or essentiallyfully clotted. The plasma can be clotted with calcium, thrombin or otherknown clotting agents, and the clotting, when performed on platelet-richplasma, can form a platelet-rich plasma gel. One skilled in the art canreadily determine appropriate amounts of clotting agents and suitableconditions for clotting.

In some embodiments, the blood-derived plastic articles arebiocompatible with the subject upon which the article is intended to becontacted. The term “biocompatible” refers to the absence of stimulationof a severe, long-lived or escalating contrary or adverse biologicalresponse to an implant or coating, and is distinguished from a mild,transient inflammation which typically accompanies surgery orimplantation of an acceptable biocompatible material into a livingorganism. Examples of suitable subjects that can be treated according tothe methods of the present invention include mammals, such as humans ordogs, and other non-mammalian animals.

In some embodiments, the blood-derived plastic articles can bebiodegradable or bioerodible, i.e., degradable in response to thesubject tissues' proteolytic processes. As used herein, “biodegradable”and “bioerodible” refer to the dissolution of a substance, such asimplant or coating, into constituent parts that may be metabolized orexcreted, under the conditions normally present in a living tissue. Insome embodiments, the rate and/or extent of biodegradation or bioerosioncan be controlled in a predictable manner.

In some embodiments, the present invention provides blood-derivedplastic articles comprising at least one (one or more) biologicalresponse modifiers. The biological response modifier(s) can be presentin the blood used to prepare the article, added to the blood and othercomponents of the composition prior to or during formation of theplastic article, and/or the plastic article can be post-treated with abiological response modifier, for example by coating with or immersioninto a composition comprising the biological response modifier(s).

As used herein, “biological response modifier” means any protein,glycoprotein, sugar, polysaccharide, lipid, DNA, RNA, aptamer, peptide,hormone, vitamin and other such substance, which when introduced into asubject is capable of eliciting a biological response, and includeshormones, cytokines, growth factors, steroids, genes, geneticallymodified organisms, such as viruses and bacteria, extracellular matrixmolecules and the like, and mixtures thereof. The term “hormone” refersto any molecule which acts as a biochemical messenger that regulatesphysiological events in living organisms, and includes growth factorsand cytokines.

Examples of suitable biological response modifiers include interleukins(IL), such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, isoforms thereof and others;interferons such as interferon alpha, beta, gamma and others; growthfactors, such as platelet derived growth factors (PDGF), acidic andbasic fibroblast growth factors including FGF-1 and FGF-2,transformation growth factors beta (TGF-beta, e.g. TGF-beta-1,TGF-beta-2 and TGF-beta-3), insulin like growth factors (IGF, e.g.,including IGF-I and IGF-II), epidermal growth factors (EGF, e.g., EGFand heparin binding EGF), platelet-derived angiogenesis factors (PDAF),platelet-derived endothelial growth factors (PDEGF), tumor necrosisfactor-alpha (TNF-.alpha.), tumor necrosis factor-beta (TNF-.beta.),vascular endothelial growth factors (VEGF), epithelial cell growthfactors (ECGF), granulocyte-colony stimulating factors (G-CSF),granulocyte-macrophage colony stimulating factors (GM-CSF), nerve growthfactors (NGF), neurotrophins, erythropoietin (EPO), thrombopoietin(TPO), myostatin (GDF-8), growth differentiation factor-9 (GDF9),hepatocyte growth factors (HGF), platelet factors, isoforms thereof,etc.; antibodies; bone morphogenetic proteins (BMPs), such as BMP-2,BMP-4, and BMP-7; extracellular matrix molecule such as osteocalcin,osteonectin, fibrinogen, vitronectin, fibronectin, thrombospondin 1(TSP-1), and bone sialoprotein (BSP), proteoglycans; metalloproteases orprometalloproteases and inhibitors thereof; angiotensin convertingenzyme inhibitors; plasminogen and tissue plasminogen activators (TPA),including anisoylated plasminogen activator (TPA) and anisoylatedplasminogen-streptokinase activator complex (APSAC) and inhibitorsthereof; xymogens such as prothrombin, plasminogen, prokallikrien,proelastase, and procollagenase; proteases such as thrombin, plasmin,kallikrien, elastase, and collagenases; protease inhibitors such asaprotinin, alpha 1-antitrypsin, alpha 2-microglobulin, alpha2-antiplasmin, anti-thrombin and tissue inhibitor of metalloproteases(TIMP1); RNA and DNA in its various forms to modify gene expression andfunction; cytokines including chemotactic cytokines (chemokine),protein-based hormones such as parathyroid hormone, engineered hormones,steroid-based hormones, such as estrogen, pregnenolone, aldosterone,estradiol, cortisol, testosterone, progesterone, etc.; peptide hormones,such as insulin, parathyroid hormone related peptide, luteinizinghormone (LH), adrenocorticotropic hormone (ACTH), follicle stimulatinghormone (FSH), and angiotensin II/III; synthetic steroids including, butnot limited to, glucocorticoids, such as prednisone, dexamethasone,triamcinolone, etc., mineralocorticoids, such as fludrocortisone,Vitamin D derivatives, such as dihydrotachysterol, synthetic androgens,such as oxandrolone, decadurabolin, etc., synthetic estrogens such asdiethylstilbestrol (DES); synthetic progestins, such as norethindroneand medroxyprogesterone acetate; and mixtures thereof. Further examplesof suitable growth factors and bone morphogenetic proteins fromplatelets are discussed in Eppley, B. L. et al., “Platelet-Rich Plasma:A Review of Biology and Applications in Plastic Surgery”, Plast.Reconstr. Surg. 118(6) pp. 147e-159e (2006) and Sipe J. B., et al.,“Localization of Bone Morphogenetic Proteins (BMPs)-2, -4, and -6 withinMegakaryocytes and Platelets”, Bone 35(6). pp. 1316-22 (2004),incorporated by reference herein.

In some embodiments, the biological response modifier is a bioactiveprotein selected from the group consisting of hormones, growth factors,cytokines, extracellular matrix molecules and mixtures thereof, such asare discussed above.

As discussed above, the biological response modifier(s) can be presentin the blood used to prepare the composition, or added to the othercomponents of the composition as a separate component prior to formationof the article, or added by post-treatment. In some embodiments, theamount of biological response modifier present in the blood orcomposition can range from about 1 picogram per gram of composition toabout 20 milligrams per gram of composition, or about 1 nanogram pergram of composition to about 1 milligram per gram of composition, orabout 1 microgram per gram of composition to about 1 milligram per gramof composition. The presence and/or amount of biological responsemodifier present in the blood or composition can be determined by assaysand analytical methods well known to those skilled in the art, forexample immunoassays including visual or fluorescent-based assays suchas ELISA (enzyme-linked immunosorbent assay) and radioactive-basedassays such as RIA (radioimmunological assay) or IRMA (immunoradiometricassay).

In some embodiments, the biological response modifier is biologicallyactive when present in the article, i.e., present before or afterformation of the article for example by compounding or plasticizing. Thelevel of biological activity can be reduced during the formation of thearticle, however, at least some biological activity is preferablyretained.

In some embodiments, the amount of biological response modifier presentin the article can range from about 1 picogram per gram of article toabout 20 milligrams per gram of article, or about 1 nanogram per gram ofarticle to about 1 milligram per gram of article, or about 1 microgramper gram of article to about 1 milligram per gram of article.

The presence and/or amount of biological response modifier present inthe blood, composition or article can be determined by assays andanalytical methods well known to those skilled in the art, such as arediscussed below. Various proliferation assays, commonly known andstandardized in the art, can be used to determine cell proliferationresponses. For example, a radiological proliferation assay can be usedfor determination of biological activity of articles or compositions ofthe present invention. Cells (NIH 3T3 mouse fibroblasts, for example)are seeded into wells of a 24 well cell culture plate at 20,000 cellsper well in serum media. Twenty four hours later the growth media isreplaced by serum-free media. Twenty four hours later the serum-freemedia is replaced with serum-free media conditioned by soaking withblood, composition or crushed blood-based biopolymer for 24 hours.Positive controls consist of cells grown in serum media and negativecontrols of cells grown in serum-free media. Twenty four hours later 2.5μCi of tritiated thymidine is added per well and four hours later thecells are washed and the incorporated thymidine isolated and countedusing protocols known in the art. To determine blood-based biopolymerbioactivity resulting in cellular proliferation over the negativecontrol, statistical analyses can be performed using multiple analysisof variance (ANOVA) and Tukey's post-hoc test for multiple comparisonanalysis, with a significance level of p<0.05.

The presence of growth factors, among other bioactive factors, within asample can be detected through the use of a variety of commerciallyavailable immunoassays, such as RIA, ELISA or IRMA. These assays areavailable for a large number of human bioactive proteins such as VEGF,BMP-2, TGF-beta, and PDGF, for example.

Various differentiation assays commonly known and standardized in theart, such as gene expression-based assays or protein expression-basedassays, can be used to determine the bioactivity of certain hormones(including cytokines, growth factors, etc.) to induce cellulardifferentiation. For example, BMP-2 will induce C2C12 mouse progenitorcells to differentiate into bone cells, as evidenced by the induction ofC2C12 alkaline phosphatase expression. C2C12 cells can be seeded ontoplasma-based biopolymers (5 mm.times.5 mm in area) with 2 mL of 60,000cells/mL in a 12 well plate in serum media. Cells seeded into an emptywell with serum media and cells in an empty well with serum media plus100 ng/mL BMP-2 serve as negative and positive controls, respectively.Forty eight hours post-seeding, the cells are stained for alkalinephosphatase using a commercial kit. Alternatively to seeding ontobiopolymer plastics, cells can be incubated in serum-free mediaconditioned by soaking with crushed plasma-based biopolymer for 24hours.

In some embodiments, the biological response modifier is heat-sensitive.As used herein, “heat-sensitive” means any compound which when heatedbeyond 50° C. becomes biologically inactive. Thus, the term“heat-sensitive” compound encompasses any compound, such as biologicalresponse modifiers, antigens, drugs, hormones, tracers, labeledcompounds, which lose biological activity at a temperature greater thanabout 50° C., or greater than about 80° C., by any means includingmelting, decomposition, denaturation, etc. In some embodiments, lowtemperature processing of the composition to form the plastic articlecan be conducted at a temperature of less than about 50° C., or lessthan about 65° C., or less than about 80° C., or about 55° C. to about65° C., or about 60° C.

In some embodiments, the composition from which the article is preparedcan further comprise at least one crosslinking agent for crosslinking orgelling various crosslinkable groups of the blood and/or with othercomponents of the composition. Suitable cross-linking agents may bephysical or chemical. Examples of suitable chemical crosslinking agentsinclude iridoid derivatives (such as genipin(Methyl(1R,2R,6S)-2-hydroxy-9-(hydroxymethyl)-3-oxabicyclo[4.3.0]nona-4,8-diene-5-carboxylate),diimidates, diones (e.g., 2,5-hexanedione), carbodiimides, (e.g.,1-ethyl-[3-(dimethylaminopropyl)]carbodiimide) (abbr., EDC), acrylamides(e.g., N,N′methylenebisacrylamide), sugars (e.g., ribose and fructose),proteins (e.g., enzymes, such as transglutaminase Factor XIII),dimethylsuberimidates, aldehydes (e.g., glutaraldehyde, andformaldehyde, formaldehyde sodium bisulfite), dihomo bifuntional NHSesters (e.g., di NHS-esters of dicarboxylic acid comprising 1-20intervening carbons), carbonyldiimide; glyoxyls; proanthocyanadin,reuterin (2-hydroxy-propanal), similar cross-linking agents and mixturesthereof.

Chemical cross-linking agents can be solids (e.g., powders) or liquids.Examples of cross-linking agents which are solids include, genipin,dihomo bifuntional NHS esters, and formaldehyde sodium bisulfite.Examples of liquid crosslinking agents include formaldehyde,glutaraldehyde, etc. In some embodiments, the article comprisesmonoaldehyde or polyaldehyde cross-linked amines; and/or pyrancross-linked amines. As used herein “cross-linked amine” refers to anybridging bond between two polymers comprising nitrogen, such as theproduct of aldehyde cross-linking (i.e., an imine or an eneamine), orproduct of an ester cross-link, or an amide, or any other similar bond.Physical cross-linking agents include, for example, electromagneticradiation, such as ultraviolet light, heat, microwaves, etc.Cross-linking may occur before or after formation of the article.Mixtures of any of the above crosslinking agents can be used.

In some embodiments, the crosslinking agents are selected from the groupconsisting of iridoid derivatives, diimidates, diones, carbodiimides,acrylamides, sugars, proteins that are chemically different from thebioactive protein, dimethylsuberimidates, aldehydes, Factor XIII, dihomobifunctional NHS esters, carbonyldiimide, glyoxyls, proanthocyanadin,reuterin, dimethylsuberimide and mixtures thereof.

Solid cross-linking agents can be active, even prior to hydration, forexample, where the polymer contains residual water and/or amino groups.Thus, where a solid crosslinking agent is used, it can be active priorto hydration or, in the alternative, upon exposure to water. Forexample, in some embodiments the solid cross-linking agent, genipin, isincorporated into the polymer admixture and subsequently allowed to beactivated by water which is either already in the polymer, blood orcomposition and/or which is absorbed when the subsequently formedpolymer matrix is in a hydrated liquid environment. Because water iseither already in the matrix (e.g., because of residual water in thematrix) or diffuses into a polymer matrix (e.g., from immersion into ahydrated environment), cross-linking can occur throughout the material.This method obviates the problem often observed with liquid crosslinkingagents, which cross-link as they diffuse into the gel, creating a stiffouter shell, while the internal part of the gel swells with water sinceit has not been exposed to the cross-linking agent due to the slowdiffusion of the cross-linking agent. Such inhomogeneity can createpressure within the structure, sometimes leading to cracking anddeformation. Thus in some embodiments, incorporation of the crosslinkerinto the composition as a solid, such as genipin, can be used. Genipinis a pyran hydrolytic product of geniposide, and is capable of formingcross-links with amines and with itself. Genipin is a cross-linkingagent of low toxicity, and, thus, better suited for use in numerousbiomedical applications, since many other cross-linking agents such asglutaraldehyde can be toxic to cells. In addition, genipin conjugatescan turn a brown and/or a blue color and fluoresce, thus allowing visualmonitoring of the extent and positions of the cross-linking, inreal-time.

Genipin can be added as a powder up to about 2% by weight or more of thedried blood weight, prior to dough mixing and plasticizing. Genipinpowder (known in the art) can be solubilized in alcohol, such asethanol, methanol, glycerol, isopropanol, propylene glycol, or any ofthe di-, tri- or tetra-polyethylene glycols. Also, since an alcohol canbe used to sterilize the plasma powder, genipin or other crosslinkingagents may be added to the alcohol during sterilization, and can beretained in the bioplastic dough while the alcohol fraction is removed.It also should be understood that genipin in solution can be admixedinto the bioplastic dough, or genipin in solution can be infused into aplasma gel, but it may also be incorporated as a dry powder with any ofthe present bioplastic ingredients at any step of processing. Othercrosslinkers, both water- and alcohol-soluble, known in the art may besubstituted.

The amount of crosslinking agent used in the composition can range fromabout 0.01 to about 20 weight percent, or about 0.1 to about 20 weightpercent, or about 0.1 to about 10 weight percent, or about 0.1 to about1 weight percent, on a basis of total weight of the composition.

As used herein, the term “cross-link” or “crosslink” as used inconnection with a composition, means that any crosslinking agent in thecomposition has at least partially reacted with itself and/or withfunctional groups of components of the composition, creating crosslinkstherein. In some embodiments, the degree of crosslinking, i.e., thedegree of total functional groups of the crosslinking agent that havereacted within the composition, ranges from about 50% to about 100% ofcomplete crosslinking where complete crosslinking means full reaction ofthe crosslinking agent in the composition. In other embodiments, thedegree of crosslinking ranges from about 75% to about 100% or about 90%to about 100% of full crosslinking. One skilled in the art willunderstand that the presence and degree of crosslinking, i.e., thecrosslink density, can be determined by a variety of methods, such asFourier Transform Infrared Spectroscopy (FTIR), mechanical testing, gelchromatography, etc.

As used herein, the term “cure” as used in connection with acomposition, e.g., “composition when cured” or a “cured composition”,refers to the toughening or hardening of the composition, brought aboutby physical or chemical means, such as by chemical components of thecomposition having reactive functional groups, ultraviolet radiation,electron beam (EB), heat, and/or pressure. After reaction of most of thereactive groups occurs within a composition subjected to curingconditions, the rate of reaction of the remaining unreacted reactivegroups becomes progressively slower. In some embodiments, the curablecomposition can be subjected to curing conditions until it is at leastpartially cured. The term “at least partially cured” means subjectingthe curable composition to curing conditions, wherein reaction of atleast a portion of the reactive groups of the composition occurs, toform a partially cured composition. In some embodiments, the compositioncan be subjected to curing conditions such that a substantially completecure is attained and wherein further exposure to curing conditionsresults in no significant further improvement in properties, such asstrength or hardness.

The articles of the present invention can be in the form of a hydrogelprior to application to a subject. A “hydrogel” is defined as asubstance formed when a polymer (natural or synthetic) becomes a 3-Dopen-lattice structure that entraps solution molecules, typically water,to form a gel. A polymer may form a hydrogel by, for example,aggregation, coagulation, hydrophobic interactions, cross-linking, saltbridges, etc. A plasma gel is a hydrogel classically formed by clottingmethods well known in the art (e.g., by adding thrombin, calciumchloride, etc.). Alternatively, plasma can be formed into a hydrogelthrough the addition of other exogenous factors, such as crosslinkers.Where a hydrogel is to be used as part of a scaffold onto which cellswill be seeded, the hydrogel should be non-toxic to the cells. The term“dehydrated” whether referring to a structure, such as a film, or ahydrogel includes any substance that has had water removed from it byany processes, and, thus, includes partially hydrated hydrogels, such asthose described herein.

A “hydrogel solution” is a solute and a solvent comprising a substancethat if subjected to the appropriate conditions, such as temperature,salt concentration, pH, the presence of a protease, the presence of abinding partner, etc., becomes a hydrogel or part of a hydrogel. Theterm “solution” in a hydrogel solution is intended to include truesolutions, as well as suspensions, such as colloidal suspensions, andother fluid materials where one component is not truly solubilized.

In some embodiments, the present invention provides blood-derivedplastic articles prepared from a composition comprising: (1) blood(which optionally can comprise one or more biological response modifiersand/or other components discussed below) and (2) at least onecrosslinking agent selected from the group consisting of iridoidderivatives, diimidates, diones, carbodiimides, acrylamides, sugars,proteins that are chemically different from the bioactive secretoryprotein, dimethylsuberimidates, aldehydes, Factor XIII, dihomobifunctional NHS esters, carbonyldiimide, glyoxyls, dimethylsuberimide,proanthocyanadin, reuterin, and mixtures thereof. Suitable bloodproducts and crosslinking agents and amounts of the same are discussedin detail above.

Any of the compositions discussed above can further comprise at leastone plasticizer in addition to any plasticizer (such as water) in theblood. Examples of suitable plasticizers include phthalate plasticizers,adipate plasticizers, trimellitate plasticizers, maleate plasticizers,sebacate plasticizers, benzoate plasticizers, plant oils, such asepoxidized vegetable oils, animal oils, mineral oils, sulfonamideplasticizers, phosphate plasticizers, water, polyalcohols, glycols,glycerol (glycerin), polyethers, acetylated monoglycerides, alkylcitrates, polymeric plasticizers and functionalized derivatives thereof,such as poly(ethylene glycol) diacrylate, and mixtures thereof. In someembodiments, the plasticizer can have reactive functional groups whichare capable of polymerizing with itself, the blood and/or othercomposition components during mixing and/or curing of the composition.In some embodiments, the plasticizer is glycerol and/or water.

The amount of plasticizer used in the composition can range from about0.1 to about 80 weight percent, or about 5 to about 60 weight percent,or about 10 to about 60 weight percent, or about 20 to about 50 weightpercent, on a basis of total weight of the composition.

In some embodiments, the composition further comprises at least onedrug. The term “drug” refers to a substance used as a medication or inthe preparation of medication, including, but not limited to, asubstance intended for use in the diagnosis, cure, mitigation,treatment, or prevention of a condition, such as infection, disease, ortrauma. For example, a drug may include, but is not limited to, smallorganic molecules, complex organic molecules, inorganic elements andmolecules, and the like. As used herein, the term “drug” encompasses forexample, fungicides, anticoagulants, antibiotics, antivirals,anti-inflammatories, both steroidal and non-steroidal, antibodies, andother molecules. Examples of suitable drugs include analgesics;anti-infective agents such as antibiotics (for example cephalosporins;penicillins; aminoglycosides including gentamicin and neomycin;glycopeptides including vancomycin; macrolides including azithromycinand clarithromycin; quinolones including ciprofloxacin, gatifloxacin,and levofloxacin; sulfonamides; and tetracycline), antifungals (forexample polyene antifungals, imidazole antifungals and triazoleantifungals), and antivirals; antineoplastics such as antibiotics,antimetabolites, hormonal agonists/antagonists, androgens,immunomodulators, skin and mucous membrane agents and steroids;biologicals; blood modifiers such as anticoagulants, antiplateletagents, colony stimulating factors, hematinics, hemorrheologic agents,hemostatics, thrombin inhibitors and thrombolytic agents;cardioprotective agents; cardiovascular agents such as adrenergicblockers, adrenergic stimulants, angiotensin converting enzyme (ACE)inhibitors, angiotensin II receptor antagonists, antiarrhythmics,antilipemic agents, beta adrenergic blocking agents, vasodilators, andvasopressors; cholinesterase inhibitors; hormones such as: anabolicsteroids, androgens, estrogens and combinations, glucocorticoids andgrowth hormone; immunomodulators; immunosuppressives; ophthalmicpreparations such as antibiotics, anti-infectives, anti-inflammatoryagents and beta adrenergic blocking agents; respiratory agents such asanti-infective agents, anti-inflammatory agents, skin and mucousmembrane agents such as analgesics, anti-infectives, antibiotics,antifungals, antivirals, antineoplastics, anti-cancer agents andmixtures thereof.

The drug can be administered via the article in a “therapeuticallyeffective amount”, i.e., that amount of a pharmacological or therapeuticagent that will elicit a biological or medical response of a tissue,system, or subject that is being sought by the administrator (such as aresearcher, physician, clinician or veterinarian) which includesalleviation of the symptoms of the condition or disease being treatedand the prevention, slowing or halting of progression of the condition,including but not limited to infection, disease or trauma.

The amount of drug used in the article can range from about 0.001 toabout 10 weight percent, or about 0.001 to about 5 weight percent, orabout 0.001 to about 1 weight percent, on a basis of total weight of thecomposition. The amount of drug used in the composition to prepare thearticle can be the same as is desired in the article or higher toaccount for loss of activity (if any) during preparation of the article.One skilled in the art can determine the amount of desired drug byroutine experimentation, or for example the amount of drug used in thecomposition can range from about 0.001 to about 10 weight percent, orabout 0.001 to about 5 weight percent, or about 0.001 to about 1 weightpercent, on a basis of total weight of the composition. Alternatively oradditionally, one or more drugs can be included for co-administration ordelivery with the article, for example by coating or impregnating atleast a portion of the article.

In some embodiments, the composition further comprises at least onestabilizer. The stabilizer may be added to the plasma constituents toprotect endogenous plasma proteins during dehydration, rehydration,lyophilization and/or subsequent milling. Examples of suitablestabilizers include glycogen, sorbitol, mannitol, trehalose, maltitol,xylitol, isomaltitol, erythritol, amylose, amylopectin, inositolhexasulfate, sulfated beta-cyclodextran, betaine, nontoxicpolysaccharide according to the general formula of C_(n)(H₂O)_(n-1)where n is between 200 and 2500, antioxidants, and mixtures thereof.

The amount of stabilizer used in the composition can range from about0.1 to about 70 weight percent, or about 0.1 to about 25 weight percent,or about 0.1 to about 10 weight percent, on a basis of total weight ofthe composition.

In some embodiments, the composition can further comprise at least onefiller. Examples of suitable fillers include any substance incorporatedinto the polymer in order to provide additional structural or mechanicalproperties to the compositions disclosed herein, for exampleparticulates such as calcium phosphate, tricalcium phosphate, calciumsulfate, hydroxyapatite, excipients (e.g., inert compounds acting asbulking agents, such as carboxymethylcellulose or starch), syntheticand/or naturally occurring substances, such as polysaccharides andproteins (e.g., fibrous or globular proteins), which can be, forexample, inert or biologically active or inactive, and mixtures thereof.In some embodiments, the fillers can be nanoparticulates.

The amount of filler used in the composition can range from about 0.1 toabout 75 weight percent, or about 5 to about 70 weight percent, or about25 to about 60 weight percent, on a basis of total weight of thecomposition.

In some embodiments, the composition further comprises at least oneporogen. The term “porogen” refers to any particulate incorporated intoa polymer matrix, wherein the particulate can be removed by any meansincluding dissolution or sublimation of the porogen into a liquid or gasphase. A porogen can be soluble in the aqueous phase, the organic phase,or capable of sublimation into a gas. A porogen can also comprise anencapsulated gas (i.e., CO₂, nitrogen, oxygen, etc.) or substancecapable of releasing a gas, upon decomposition, such as, for example,sodium bicarbonate releasing CO₂ upon contact with an acid. Examples ofsuitable porogens include polyurethane, polylactic acid, polyglycolicacid, polylactic-co-glycolic acid, and polycaprolactone; a porogensoluble in an aqueous phase, such as sodium chloride; or a sublimationporogen, such as ammonium acetate, ammonium chloride, ammonium sulfate,ammonium bicarbonate, ammonium carbonate or pyridinium trifluoroacetate,and mixtures thereof. In some embodiments, the introduction ofparticulate ammonium acetate crystals, pre-sized to 150-250 microns,during the dough mixing phase, and following sublimation (drying undervacuum) post processing, resulted in a controlled porous plastic with apore size of 150-250 microns.

The amount of porogen used in the composition can range from about 0.1to about 95 weight percent, or about 20 to about 90 weight percent, orabout 30 to about 75 weight percent, on a basis of total weight of thecomposition.

In some embodiments, the composition further comprises at least onepolymeric material, including biocompatible polymeric materials such aspolymeric sugars, for example polysaccharides (e.g., chitosan) andglycosaminoglycans, (e.g., hyaluronan, chondroitin sulfate, dermatansulfate, keratin sulfate, heparan sulfate, and heparin), polymericproteins, such as fibrin, collagen, fibronectin, laminin, and gelatin,and mixtures thereof. Examples of biocompatible, non-biodegradablepolymers include, but are not limited to, polyethylenes, polyvinylchlorides, polyamides, such as nylons, polyesters, rayons,polypropylenes, polyacrylonitriles, acrylics, polyisoprenes,polybutadienes and polybutadiene-polyisoprene copolymers, neoprenes andnitrile rubbers, polyisobutylenes, olefinic rubbers, such asethylene-propylene rubbers, ethylene-propylene-diene monomer rubbers,and polyurethane elastomers, silicone rubbers, fluoroelastomers andfluorosilicone rubbers, homopolymers and copolymers of vinyl acetates,such as ethylene vinyl acetate copolymer, homopolymers and copolymers ofacrylates, such as polymethylmethacrylate, polyethylmethacrylate,polymethacrylate, ethylene glycol dimethacrylate, ethylenedimethacrylate and hydroxymethyl methacrylate, polyvinylpyrrolidones,polyacrylonitrile butadienes, polycarbonates, polyamides,fluoropolymers, such as polytetrafluoroethylene and polyvinyl fluoride,polystyrenes, homopolymers and copolymers of styrene acrylonitrile,cellulose acetates, homopolymers and copolymers of acrylonitrilebutadiene styrene, polymethylpentenes, polysulfones, polyesters,polyimides, polyisobutylenes, polymethylstyrenes, other similarcompounds known to those skilled in the art, and mixtures thereof. Otherbiocompatible non-degradable polymers that are useful in accordance withthe present invention include polymers comprising biocompatible metalions or ionic coatings which can interact with DNA, for example gold andsilver ions may be used for inhibiting inflammation, binding DNA, andinhibiting infection and thrombosis. Examples of biocompatible,biodegradable polymers include, but are not limited to, polylactic acid(PLA), polyglycolic acid (PGA), polylactic-co-glycolytic acid (PLGA),polycaprolactone, and copolymers thereof, polyesters, such aspolyglycolides, polyanhydrides, polyacrylates, polyalkyl cyanoacrylates,such as n-butyl cyanoacrylate and isopropyl cyanoacrylate,polyacrylamides, polyorthoesters, polyphosphazenes, polypeptides,polyurethanes, polystyrenes, polystyrene sulfonic acid, polystyrenecarboxylic acid, polyalkylene oxides, alginates, agaroses, dextrins,dextrans, and polyanhydrides. Mixtures of any of the above polymericmaterials can be used.

The amount of polymeric material used in the composition can range fromabout 0.1 to about 90 weight percent, or about 10 to about 80 weightpercent, or about 15 to about 75 weight percent, on a basis of totalweight of the composition.

In some embodiments, the composition further comprises at least onetracer, labeled compound or mixtures thereof. The term “tracer” refersto any molecule that is introduced into an organism or construct andcapable of being detected. For example, tracers include, but are notlimited to, radioactive compounds, contrast agents, light-emittingmolecules, quantum dots, fluorescent molecules, dyes, biomarkers,molecular tracers for imaging purposes (including fluorescence markers,radioactive markers, contrast agents for CT, microCT, MRI or forms ofbio-imaging, and immunospecific markers), and others. As used herein, a“labeled compound” refers to any substance modified such that it (or itsmetabolites, such as degradation products) is detectable by any means. Alabeled compound may be labeled in any manner including attachment(e.g., covalent or non-covalent) of tracers to the molecule of interest.

In some embodiments, the composition can further comprise metal ions,for example gold and silver ions can be used for inhibitinginflammation, infection and/or thrombosis. The amount of metal ions usedin the composition can range from about 0.01 to about 5 weight percenton a basis of total weight of the composition.

In some embodiments, the present invention provides methods of makingblood-derived plastics using blood which is at least partially clottedeither before or after removal of any desired constituents, at leastpartially dried and optionally powdered, mixed with other componentssuch as plasticizer, etc. as discussed above, and processed into aplastic article.

Suitable methods for clotting the blood, such as admixing with calcium,thrombin or another suitable clotting agent, are discussed above. Theclotted blood can be at least partially dried to a water content asdescribed above by a variety of methods, such as freeze drying(lyophilization), heating in a conventional oven, air-drying, etc. Insome embodiments, the clotted blood is lyophilized at a temperature ofless than about −18° C. In some embodiments, the clotted blood is driedat a temperature of less than about 50° C., or less than about 80° C.

In some embodiments, the clotted and dried blood can be ground into apowder or otherwise comminuted by any means known in the art, forexample by milling, grinding, spray-drying, etc. In some embodiments,the average diameter of the blood powder particles can be less thanabout 30 mesh (i.e., about 595 microns), less than about 35 mesh (i.e.,about 500 microns), less than about 100 mesh (i.e., about 149 microns),less than about 200 mesh (i.e., about 74 microns), or less than about400 mesh (i.e., about 37 microns) or about 10 to about 800 microns.

Prior to further processing, the dried blood or powder can be treated(washed) with ethanol or propanol to sterilize it and, if desired, toremove unwanted salts from the blood by removing the wash-step alcohol.

In some embodiments, the clotted and dried blood (optionally powdered)can be mixed with other components of the composition in amounts asdescribed above, such as plasticizer and/or crosslinking agent, in anyconventional manner, for example by mixing in a container using astainless steel stirrer for about 2 minutes to about 24 hours at atemperature of about 25° C.

In some embodiments in which a solid crosslinking agent, such asgenipin, is used, the crosslinking agent can be pre-dissolved in asolvent prior to mixing with the other components of the composition.Examples of suitable solvents include alcohols such as ethanol orisopropanol. The amount of solvent used can be that amount which issufficient to at least partially or fully dissolve the solidcrosslinking agent, for example about 1 to about 200 mg of crosslinkingagent per ml of solvent.

In some embodiments, the clotted and dried blood may be added tovirtually any polymeric or plastic base material that will cure at thedesired temperatures. In some embodiments, clotted and dried plasma,plus plasticizer (water and/or glycerol) is used to make a bioplasticmaterial without other structural-plastic-making additives. Except forconstituting materials such as powders, additives, biologics or drugs,etc., in some embodiments the present inventive compositions consistessentially of clotted and dried plasma plus plasticizer.

The composition can be formed into a plastic article by any suitablemeans known in the art, for example by molding, extrusion, casting orprinting. In some embodiments, articles can be formed by extruding theplastic precursors through a die so that the extruded plastic is shapedas desired, for example in the shape of a film, sheet, tube, filament,rod or sheet. Extrusion can be accomplished at relatively low pressuresand temperatures; under certain processing conditions the plastic may bepartially or completely dehydrated by the extrusion process. Aftersufficient dehydration, with or without the use of osmotic membranesand/or lyophilization, the extruded material may be plasticized and,optionally, cross-linked. In some embodiments, extrusion can create analignment, i.e., anisotropy, of the constituent molecules within theplastic and so impart certain properties, such as toughness, to thefinal elastomeric and/or pliant materials. In some embodiments,biological response modifiers, drugs, antigens, tracers, or other suchmolecules can be included in the composition or added into the bulkplastic material, such as the admixture or slurry, prior to processing.Suitable extrusion equipment is well known to those skilled in the art,for example Brabender extrusion equipment. The processing temperatureused in the extruder can be uniform or vary across the extrusion zones,as desired. Examples of suitable processing temperatures can be lessthan about 50° C., or less than about 80° C., to preserve biologicalactivity. Higher processing temperatures can be used, if desired.

In some embodiments, articles can be formed by molding, for example bycompression molding. Suitable molding apparatus are well known to thoseskilled in the art. In some embodiments, the molding process can beconducted at a temperature of about 45° C. to about 150° C. at apressure of about 5 to about 50 kpsi. The molding process can includeuse of a release agent to facilitate mold release. In some embodiments,the molding apparatus can include a cooperating hot press and a vacuumdegasser.

Fabrication can also be by powder molding according to the followingalternative method. Molds are filled with powdered materials, includingthe powdered plasma, and subsequently infiltrated with plasticizer suchas glycerol under positive pressure. Similarly, negative pressure may beapplied to the bottom of the powder bed as glycerol, or otherplasticizer, is applied over the top, or a combination of both, byvacuum casting. The resulting powdered structure can be compacted bycompression molding according to PCT/US06/29754.

As an alternative to molding, powdered materials can be selectivelydeposited, voxel-by-voxel and layer-by-layer into a mold cavity to formeither homogenous or heterogenous 3D structures. Then, glycerol, oranother plasticizer, can be infused into the structure under positivepressure, or by applying a negative pressure to the bottom of the powderbed as glycerol is applied over the top, or a combination of both. Theresultant powdered structure may be compacted by compression moldingaccording to PCT/US06/29754.

In some embodiments, the water content of a composition can becontrolled by vacuum drying, i.e., by controlling vacuum and/ortemperature so as to dehydrate the composition. Such vacuum processingtechniques are especially useful for large scale processing. In someembodiments, the water content of the composition is controlled byevaporating the water under normal atmospheric pressure. Evaporativeprocesses may be performed at any temperature. In some embodiments, thetemperature used to evaporate the water is less than the temperature atwhich molecules incorporated into the polymer matrix would denature.This is referred to as the subcritical pressure and/or temperature forthe inclusion present in a polymer matrix. In some embodiments, suchprocessing techniques allow the polymer matrix to be loaded with asubstance, such as a crosslinking agent, and subsequently formed into astructure, such as a film, without loss of bioactivity of theincorporated substance. For example, a composition can be dehydrated atvarious temperatures that would prevent the degradation or denaturationof heat-sensitive chemicals and proteins, e.g., at a temperature of lessthan 80° C., less than 70° C., less than 65° C., less than 60° C., lessthan 55° C., less than 50° C., less than 45° C., less than 40° C., lessthan 35° C., less than 30° C., less than 25° C., or room temperature, orless. Use of temperatures of less than room temperature or even lessthan 4° C. are also possible, such as freeze-drying of the composition.Pressure may also be regulated during the drying process. Pressures maybe reduced below a normal atmosphere by any means, including use of agel dryer connected to a vacuum source. Vacuum pressure can be less than100 millibars, less than 50 millibars, less than 25 millibars, less than20 millibars, less than 15 millibars, less than 10 millibars, less than5 millibars, less than 1 millibar, or even less. Those of skill in theart recognize that by reducing the pressure and/or increasing thetemperature, the drying time can be decreased. Thus, drying may occurover any time period, such as over 1 hour, 2 hours, 4 hours, 8 hours, 16hours, 24 hours, or longer. Moreover, the drying time can be varied toallow the composition to remain partially hydrated; i.e., wherein notall of the trapped water in the composition is removed. In someembodiments, the composition can be dried on a substantially planarsurface, thus creating a substantially planar film, for example byplacing in a frame, and/or compressing between sheets of material thatpreserve the forms, such as plastic sheets. In some embodiments, thecomposition can be dried over a formed shape, thus creating a formedfilm that can be removed from the shape. In some embodiments, thecomposition can be dried directly onto a structure or surface and notremoved, thereby creating a film coating on the structure or surface.

In some embodiments, water and plasticizer can be added before formationof the article. In some embodiments, once the water is removed from thecomposition, plasticizer can be added to the resulting material. In someembodiments, the addition of plasticizer can be accomplished by soakingthe dehydrated material in a bath of the plasticizer. In someembodiments, tracers, and/or labeled compounds may be incorporated intothe polymer matrix.

In some embodiments, the processing steps can be performed under tensileload conditions to modify subsequent biomechanical properties of thematerial by aligning filaments of the component material, e.g., fibrin.When plasticizer is added to the resulting material the orientation ofthe components of material can exhibit improved mechanical propertiesfor application as graft substitutes for soft tissue repair includingvascular, tendon and ligament tissues.

In some embodiments, the plasticizing temperature can be between about55 to about 65° C. In some embodiments, the clotted driedblood-containing composition may be plasticized at temperatures up toabout 150° C., particularly to create harder and/or denser bioplasticmaterials. In some embodiments, the clotted dried plasma containingadmixtures of the present invention can be plasticized at suitablepressures, for example about 9 to about 25 kpsi (kilopounds per squareinch), about 9 to about 15 kpsi or at least 10.7 kpsi or higher. Theresulting plasma-based plastics (PBPs) of the present invention can thusbe made with a range of biomechanical and degradation properties. PBPscan be used in a variety of clinical applications, including their useas substitute graft materials, drug delivery carriers, anti-adhesion andbarrier membranes and scaffolds for tissue engineering. PBPs can also beused in cell culture as a non-animal source of endogenous or exogenousgrowth media.

Polymer molecular weight can be determined by gel permeationchromatography (GPC); bond structure by infrared (IR) spectroscopy; andtoxicology by initial screening tests involving Ames assays and in vitroteratogenicity assays, and implantation studies in animals forimmunogenicity, inflammation, release and degradation studies.

In some embodiments, the plastic articles are capable of deformation.Such plastics may be hard or soft plastic, depending on intended use.These polymers may be shaped, machined, formed, molded, extruded, etc.,into desirable shapes depending on the intended uses.

Further, the porosity of such articles may be modified by any number ofmethods including introduction of a porogen which may be intercalatedinto the polymer matrix until removed by such means as solvation andsublimation, for example. The hydration of polymers may be adjusted inany manner, including removal of water by evaporation, osmosis, or anyother method. Such procedures may be performed for a time, temperature,and/or pressure suitable for the intended application. Thus, in someembodiments, low temperature manufacturing processes are presented.

In some embodiments, powdered clotted blood can be used to make abioplastic, together with water and/or glycerol plasticizer. The clottedand dried plasma may alternatively be added to virtually any plasticbase material that will cure at the desired temperatures.

In some embodiments in which powdered clotted plasma is used as aningredient in bioplastics, the powder can be adjusted to a water contentof 5-15% by weight, or 8-12% by weight, or 8-10% by weight, prior tomixing the dough. By contrast, when the plasma or plasma fraction isdried through the gel phase, the water that is inherent from theoriginal plasma clot can represent about 10-25% by weight of thestarting material. Also, at any time a stabilizer may be added to theplasma to protect it during dehydration and rehydration. In someembodiments, the plasticizing temperature can be between 55-65° C. Insome embodiments, the clotted dried plasma containing composition may beplasticized at temperatures up to about 150° C.

The articles of the present invention can be formed or post-fabricatedto possess one or more desired mechanical properties related to adesired use. A “mechanical property” refers to essentially any propertythat provides some description for how a substance responds to theapplication of an external force. Exemplary mechanical propertiesinclude tensile strength, compression strength, flexural strength,impact strength, elongation, elasticity, stiffness, toughness, havingmechanical properties similar to rubber (e.g., rubbery), etc. Tensileand compression physical properties for plastics can be determined usingASTM methods D638-03 and D695-02a, and methods referenced therein,incorporated by reference herein. Such testing can be conducted using anInstron or similar test system. Impact strength can be determined usingASTM methods D256-02a and 4508-06, and methods referenced therein,incorporated by reference herein. Flexural strength can be determinedusing ASTM methods D790-07 and 6272-02, and methods referenced therein,incorporated by reference herein. Toughness can be determined using ASTMmethod D5045-99 (2007)e1, and methods referenced therein, incorporatedby reference herein.

In some embodiments, blood-derived plastic bone tissue articles areprovided having a Young's Modulus ranging from about 0.03 GPa to about50 GPa measured according to ASTM Method No. D638-03 (type V specimen, 1mm in thickness, and conditioned for 40 hours at 23+/−2° C. and 50+/−5%humidity prior to testing at 10 mm/min test speed at a pressure of about101 KPa (about 1 atm)) conducted using an Instron tester.

As directed in the ASTM, the Young's Modulus (elastic modulus) isdefined as the ratio of stress to the corresponding strain below theproportional limit of a material, in units of force per unit area.Percent strain at failure may be obtained directly from the stress vs.strain plot used to determine the Young's Modulus. Data acquired fromASTM 638-03 can also be used to derive the stiffness of a specimen, aproperty which describes the resistance of an elastic body to deflectionby an applied force. The stiffness is equal to the Young's Modulusmultiplied by the cross-sectional area of the gage-length segment of thespecimen and divided by the original grip separation distance (asdefined for sample V materials tested as directed under ASTM 638-03),and is expressed in units of force per unit distance.

These articles also have a compressive strength ranging from 1 MPa toabout 250 MPa according to ASTM No. D695-02a (cylindrical specimen ofdimensions 12.7 mm diameter×25.4 mm length, conditioned for 40 hours at23+/−2° C. and 50+/−5% humidity, and tested at 1.3 mm/min test speed ata pressure of about 101 KPa (about 1 atm)) using an Instron tester.

In some embodiments, blood-derived plastic tendon tissue articles areprovided having a Young's Modulus ranging from about 0.5 GPa to about1.5 GPa measured according to ASTM Method No. D-638-03 as discussedabove, a percent strain at failure ranging from about 8% to about 16%according to ASTM Method No. D-638-03 measured as discussed above, and astiffness ranging from about 100 N/mm to about 5000 N/mm according toASTM Method No. D-638-03 measured as discussed above, the Young'sModulus, percent strain at failure and stiffness being determined at atemperature of about 25° C. and a pressure of about 101 KPa (about 1atm).

In some embodiments, blood-derived plastic ligament tissue articles areprovided having a Young's Modulus ranging from about 100 MPa to about1000 MPa measured according to ASTM Method No. D-638-03 as discussedabove, and a stiffness ranging from about 50 N/mm to about 1000 N/mmaccording to ASTM Method No. D-638-03 measured as discussed above, theYoung's Modulus and stiffness being determined at a temperature of about25° C. and a pressure of about 101 KPa (about 1 atm).

In some embodiments, blood-derived plastic cartilage tissue articles areprovided having a Young's Modulus ranging from about 1 MPa to about 250MPa measured according to ASTM Method No. D-638-03 as discussed above, apercent strain at failure ranging from about 0.1% to about 1% accordingto ASTM Method No. D-638-03 measured as discussed above, and a stiffnessranging from about 5 N/mm to about 4000 N/mm according to ASTM MethodNo. D-638-03 measured as discussed above, the Young's Modulus, percentstrain at failure and stiffness being determined at a temperature ofabout 25° C. and a pressure of about 101 KPa (about 1 atm).

In some embodiments, blood-derived plastic skin tissue articles areprovided comprising at least one biological response modifier, whereinthe article has a Young's Modulus ranging from about 0.1 MPa to about 20MPa measured according to the “Skin Young's Modulus Test” describedbelow, and an elasticity ranging from about 50% to about 100% accordingto the Elasticity Test described below, the Young's Modulus andelasticity being determined at a temperature of about 25° C. and apressure of about 101 KPa (about 1 atm).

In some embodiments, blood-derived plastic skin tissue articles preparedfrom components comprising: (1) blood plasma and (2) at least onecrosslinking agent selected from the group consisting of iridoidderivatives, diimidates, diones, carbodiimides, acrylamides, sugars,proteins that are chemically different from the bioactive secretoryprotein, dimethylsuberimidates, aldehydes, Factor XIII, dihomobifunctional NBS esters, carbonyldiimide, glyoxyls, proanthocyanadin,reuterin, dimethylsuberimide and mixtures thereof are provided, whereinthe article has a Young's Modulus ranging from about 0.1 MPa to about 20MPa measured according to the “Skin Young's Modulus Test” describedbelow, and an elasticity ranging from about 50% to about 100% accordingto the Elasticity Test described below, the Young's Modulus andelasticity being determined at a temperature of about 25° C. and apressure of about 101 KPa (about 1 atm).

Young's Modulus and % elasticity of thin films can be measured in anumber of ways for skin, for example with suction chamber devicesdesigned for use with skin, as described by Pedersen L., et al.,“Mechanical Properties of the Skin: A Comparison Between Two Suction CupMethods”, Skin Research and Technology 9: 111-115 (2003). Briefly, inusing the DermaLab system (Cortex Technology), a small suction probe isattached to the skin (or thin film) with adhesive. The probe pullsnegative pressure to lift the skin a predetermined distance. Thesystem's software uses this information to calculate Young's Modulus.Young's Modulus testing according to this method is referred to hereinas the “Skin Young's Modulus Test”. Elasticity is determined using datacollected by the probe during both the skin elevation and retractionphases. Elasticity testing according to this method is referred toherein as the “Elasticity Test”.

Other useful mechanical properties include, for example, pliability(i.e., “pliant” is the ability of a polymer to bend or deform withoutbreaking), elasticity (i.e., “elastomeric” is the ability of a polymerto recover the original shape after deformation) and other suchproperties.

In some embodiments, the articles of the present invention are in theform of films. A film can be, for example, both elastic and pliant, orpliant without being elastic. Where a film is neither elastic norpliant, it is referred to herein as “rigid”. A “film” refers to a thinsheet. Thus, a film can be a sheet up to 1000 μm thickness, up to 100 μmthickness, up to 10 μm thickness, up to 1 μm thickness, or any rangetherebetween. A film will have many mechanical properties, such as, forexample, elasticity, non-elasticity, pliancy, rigidity, etc., dependingon the formulation and shape. In some embodiments, the film can haveless than about 5 weight percent water content on a basis of totalweight of the film, or less than about 1 weight percent water content,as desired.

In some embodiments, the articles of the present invention are in theform of powders. The term “powder” or “powdered” refers to small solidparticles. Powders, as used herein, comprise particles having an averagediameter of less than about 30 mesh (i.e., about 595 microns), less thanabout 35 mesh (i.e., about 500 microns), less than about 100 mesh (i.e.,about 149 microns), less than about 200 mesh (i.e., about 74 microns),or less than about 400 mesh (i.e., about 37 microns) or about 10 toabout 800 microns. A powder can be formed by any means known in the artor disclosed herein including milling, grinding, spray-drying, etc.

In some embodiments, the articles of the present invention are in theform of granules or agglomerates of powder particles. Granules can havean average diameter ranging from about 250 μm to about 5 mm. Granulescan be formed, for example, by agglomerating powders or by chopping,grinding or comminuting pieces or larger sized articles. In someembodiments, the granules can be molded or extruded into various shapes,such as rods for spinal fusion.

In some embodiments, the articles of the present invention can be in theform of stacked or laminated layers of films or sheets, a tubular roll,or combinations thereof.

In some embodiments, the articles of the present invention can be in theform of a bone substitutes cartilage substitute, tendon substitute,ligament substitute, skin substitute, cornea substitute, stent, fixationplate, screw, suture or staple.

In some embodiments, the articles of the present invention can havedifferent physical or chemical characteristics within the article, suchas a gradient or multiple gradients of selected characteristics.Examples of physical characteristics that can vary within the articleinclude density, porosity, elasticity and/or tensile strength. Examplesof chemical characteristics that can vary within the article includeconcentration of selected biological response modifiers and/or drugs.

In some embodiments, the article can have a density gradient from aregion of lower density to a region of higher density. In someembodiments, the article can have multiple regions of differentgradients. In some embodiments, the article can have different regionscorresponding to the tissue(s) which it is intended to replace orsupplement, for example a region having physical characteristics similarto tendon and a region having physical characteristics similar to bonewhen the article is intended to replace or supplement tendon and bone.

In some embodiments, the article can be assembled from portions havingdifferent physical or chemical characteristics, for example by stackinglayers in which one or more of the layers have differentcharacteristics. In some embodiments, articles having different regionsof porosity can be formed by assembling layers having grooves and/orperforations, filling the interstices with a porogen, then molding theassembly to evaporate the porogen and form the article. The individuallayers can be formed by any suitable method, such as extrusion ormolding.

In some embodiments, the articles of the present invention can besterilized by post-treatment, for example by exposure to radiation suchas gamma rays, or heating or steam sterilization in an autoclave toreduce or eliminate transmissible agents (such as fungi, bacteria,viruses, prions and spore forms, etc.), in a manner well known to thoseskilled in the art.

In some embodiments, the present invention provides methods ofmanufacturing autologous bioplastics by processing a patient's owndonated blood or plasma and products produced thereby. A useful methodof making such an autologous PBP is as follows. Blood can be collectedat any time, such as prior to surgery. The blood can be spun down toobtain platelet-rich plasma (PRP) and/or platelet poor plasma (PPP)and/or serum, or comparable methods such as whole blood collection orvia apheresis are used to collect plasma from the patient without havingto collect whole blood. The blood or plasma is then clotted withcalcium, thrombin or other known clotting agents to form a plasma gel.To make rubbery-to-hard plastics, the clotted blood or plasma gel can beprocessed into a powder by drying it (this can include first removingany retained serum or not, although it is also possible to use onlyserum by drying it into a powder) and then ball milling or grinding orother powdering techniques. The drying step may or may not includelyophilization, but plasma dried “through the gel phase” for use inelastomers generally should not be lyophilized if possible (see below).Alternatively, a serum-free powder can be formed by first removing serumfrom the gel by spinning and then drying and comminuting the remainingplasma. In general, then, the method can use blood, plasma or plasmafrom which one or more constituents has been removed as desired (such asserum).

Prior to further processing, the plasma powder or dried plasma gel maybe treated (washed) with ethanol or propanol to sterilize it and, ifdesired, to remove unwanted salts from the plasma by removing thewash-step alcohol. The sterilized dried blood can be mixed with one ormore of biological response modifiers, such as growth factors, drugs orother therapeutics, fillers, porogens, crosslinkers, plasticizers andstabilizers, as discussed above and then formed into a rubbery-to-hardplastic material according to methods described above and in PCT PatentApplication PCT/US06/29754, (U.S. patent application Ser. No.11/495,115). Excipients or stabilizers such as sorbitol, mannitol and/ortrehalose may be added to the blood prior to processing to protectendogenous plasma proteins during lyophilization and/or subsequentmilling. In some embodiments, the powder formation technique may includewithout limitation, jet milling, mechanical grinding/sieving, ballmilling (as mentioned above) or other forms of particulate milling. Inaddition, putty-like graft packing materials can be made by milling theplastics into pellets and mixing the pellets with self-hardening bonecements at the time of surgery.

To make elastic sheets, the clotted plasma can be processed according tomethods described in U.S. patent application Ser. No. 11/495,115. Itshould be noted that platelet-rich plasma has inherent antimicrobialproperties, and, therefore, may not require exogenous factors to beadded to produce an antimicrobial effect if such a property is desired.Alternatively, platelet-poor plasma is also useful in creating eitherautologous or allogeneic plastic implants or other patient biomaterials.

Fabricated plastics can be milled or otherwise shaped by variousapproaches including but not limited to surface texturing, cutting andgrinding. Surface textures can either be machined post-fabrication orcan be molded into place. Alternatively, defined nano- andmicro-textures can be imparted by molds used to form plastics, allowingdirect molding of surface textures during bioplastic fabrication. Suchtextures may facilitate cell adhesion and/or physically direct cellbehavior to the PBPs.

It is possible to practice the invention in an integrated system whichcan be, for example, installed in a blood bank. It should be noted thatalthough autologous or allogeneic blood can be used as a startingmaterial for a patient's own bioplastic implant the articles of thepresent invention can be used to create shelf-stable implants and othermaterials that need not be custom manufactured patient-by-patient. Inaddition, the present bioplastics can be used as interfaces betweentissues and prostheses to improve integration.

In some embodiments, a system is provided which comprises one or more ofa centrifuge, a dryer, a powder miller, disposable molds having avariety of selected or standard shapes, compression molds and acooperating hot press and a vacuum degasser. Custom molds, based onCT/MR imaging data, could also be made by using a compact CNC millingmachine, on site, or by external vendors. Compression molds made out ofdisposable, high compression strength materials, for examplepolyetheretherketone (PEEK), can eliminate the need for cleaning andsterilizing standard molds between usages. Such a system can be placedin proximity to a blood supply source, such as a blood bank, forconvenient, cost-effective and speedy preparation of plastic articlesaccording to the present invention. Of course, the system need not bepresent in a blood bank or hospital.

Referring now to FIG. 4, a schematic showing an example of thepreparation of blood-derived plastic articles from clotted, dried andpowdered plasma is provided. As shown in FIG. 4, a patient donates blood(1) which is spun down (2) into separated PRP or PPP plasma and redblood cells, and optionally the red blood cells are reinfused into thepatient (2a). The plasma is admixed with calcium, thrombin or otherclotting agent to clot the plasma (3) and to create a gel comprised ofplasma clot and serum (4). The gel is dried (5) and ground into a powderor otherwise comminuted (6). The clotted dried plasma is then blendedinto a dough with a plasticizer, such as glycerol and/or water, togetherwith adding any optional ingredients, such as biological responsemodifiers, excipients, drugs or other ingredients, and/or athermoplastic polymer additive which supplements the bioplastic matrix(7). The composited dough is packed into a compression mold (8) andplasticized at controlled, usually low, temperature, and under pressure(9), to make an article of the present bioplastic (10). Alternatively,the same dough can be extruded instead of molded, according to meansknown in the art and as described above.

In some embodiments, autologous blood from a patient or subject can beharvested and processed into a powder or plastic and stored untilneeded. Forty-five (45) L of plasma, and possibly more, may be safelyharvested by apheresis from a healthy individual every year. Taking only25 L of plasma containing 10 grams of plasma-fibrin protein/L wouldyield 250 grams plasma-fibrin protein/year. Stated differently, a literof platelet rich plasma yields 100 g solids. Considering that this yieldcan be mixed with various extenders, such as nanoparticulate calciumphosphate and plasticizers of various types, such as 1 part plasma to 3parts extender(s), this would yield 1 kilogram of plastic per year perhuman donor. Alternatively, 100 g solids plus 66 g glycerol by weightwill yield 166 g bioplastic, enough to constitute 132 cubic centimeters.The powdered plasma may be stored essentially indefinitely as alyophilized powder or as a formed plastic under the appropriateconditions. Therefore, banking of materials becomes possible for privateand/or military applications. Custom molds and compression molds, and/orextrusion, as described above, may be included.

In the event of the use of pooled plasma, precautions are taken againstdiseases including but not limited to blood-borne pathogens. The pooledor non-autologous products are useful in the event of a traumatic eventor emergency in which the patient has no opportunity to stockpile bloodor plasma in advance of a surgery or procedure. Blood banks andhospitals therefore might well find it advantageous to manufacture andstore such plastics, or their immediate components, and thereforesalvage at least a portion of blood that has been collected but isnearing the end of its shelf life.

Uses and applications of bioplastics formed with the articles of thepresent invention include, without limitation: bone grafts, includingpacking materials; tissue engineered scaffolds (to deliver stem cellssuch as embryonic, adult, autologous, allogeneic or xenogenic stemcells); fixation devices; surgical guides; scaffolds for tendon repair;prosthetic/tissue interfaces; sutures; staples; barbs; nerve guides;wound protection; and protection of dura.

In some embodiments, the articles of the present invention can be usedas a permanent or resorbable coating and/or impregnant for a substrate,such as a metal substrate or polymeric substrate. Examples of suitablesubstrates include metal matrices, such as a mesh or stent, or a polymeror polymer-coated material such as a stent or TEFLON™ fluoropolymercoated implant. In some embodiments, a blood-derived plastic article ofthe present invention can comprise a coating prepared from othermaterials, for example polymers and/or metals such as gold or silver.Coatings can be applied in any conventional manner, such as spraying ordipping. Articles or substrates can be impregnated in any conventionalmanner, such as dipping or immersion. In some embodiments, ablood-derived plastic article of the present invention can be embeddedwithin another material, for example by encasing the blood-derivedplastic article within a plastic. Coatings on the surface of theblood-derived plastic article can provide a barrier to inhibitdegradation of the article, for example by inhibiting cell proteolysisof the article.

In some embodiments, multiple layers of polymeric films are stacked atopone another. In such structures, gradients of bioactive materials and/orpores can be created by creating layers of the films each comprising thedesired amounts of the bioactive materials within or on the surface ofeach layer and then stacking the different layers as desired. Suchstructures can be created in the manner disclosed in U.S. Pat. No.6,165,486, incorporated herein by reference. Such configurations can beuseful when creating structures to fill cranial voids, for example.

In some embodiments, polymeric films are formed into sheets, tubes,rods, or filaments. Such structures can be useful as substitute orreplacements for tendon, bone, or ligament, for example, and haveapplication in long bone and non-long bone repair. Further incorporatinggrowth factors or anabolic hormones and/or drugs can improve thebiological response associated with tissue repair. Tube based structuresalso find use as tissue engineered grafts and nerve guides, for example.Also, the films can be used as barrier membranes to protect tissues andprevent tissue adhesion. The blood-plasma derived plastic articlesdisclosed herein offer significant advantages in promoting tissue andwound repair. Methods of forming the structure, such as tubularstructures, include creating compositions which are then cast intomolds. Water can then be removed from the composition (e.g., afterremoving the tubular structure from the mold or by using osmoticmembranes as surfaces of the mold) and replaced by plasticizer. In someembodiments, sheets of the compositions disclosed herein are rolled,such as on a mandrel, to create hollow tubular structures. In someembodiments, if it is desired to have a cylindrical, non-hollowcross-section, a substantially planar composition as disclosed hereinmay be rolled up on itself. In some embodiments, once the cylindricalform has been attained the elastomer can be cross-linked to retain thetubular shape and/or stapled, heated to a fusing temperature, orotherwise held in the tubular configuration. In some embodiments,articles can be fused together to form composite articles having atleast two portions having different physical or chemical properties.

In some embodiments, the integrity of a structure, such as thosedisclosed herein can be increased by including a biocompatible mesh,such as titanium, NYLO™, or DACRON™. The mesh can be added as a layer ofthe substantially planar material prior to rolling it into a tubularstructure or it can form the outer layer of the tubular structure. Insome embodiments, the materials disclosed herein can be print, cast, orextruded onto the biocompatible mesh materials. Those of skill in theart recognize that the use of mesh materials can also be used toincrease the structural integrity of configurations other than tubularor cylindrical shapes.

In some embodiments, the article may be fabricated for short-term,long-term or permanent implantation into a subject. For example, a graftmay be used to repair or replace diseased or damaged tissue or portionsof an organ (e.g., liver, bone, heart, etc.). In some embodiments, thearticle can be biodegradable to form temporary structures. For example,a bone fracture may be temporarily repaired with a biodegradable articlethat will undergo controlled biodegradation occurring concomitantly withbioremodeling by the host's cells. In some embodiments, the article canfurther comprise less degradable materials to provide more permanentgrafts or replacements.

In some embodiments, the methods and apparatus disclosed herein may beused to create structures with specific microstructural organizationsuch that the structure has the anatomical and biomechanical features ofnaturally occurring tissues, or engineering designs that arebiologically inspired.

In some embodiments, compositions comprising heat-sensitive proteins canbe compressed at temperatures below the denaturation temperature ormelting point of the protein, thus preserving bioactivity in the polymermatrix after compression. Pressed biopolymers may be made in any shapeincluding 3-dimensional structures and 2-dimensional structures, such assheets, rods, and filaments. Biopolymer structures can be prepared usingdifferent approaches (e.g., printing, casting, cold-pressing, injectionsmolding, die extrusion etc.), wherein the amount of pressure appliedcontrols the thickness and density of the biopolymer structure. Those ofskill in the art recognize that compression may be accomplished by anymeans including, for example, using a pellet press. Compression mayoccur at any suitable combination of pressure and temperature, such asare discussed above. In some embodiments, a mold release agent, such aslecithin, is used to facilitate removal of an article from a press ormold. In some embodiments, the mold temperature is decreased from aninitial value of approximately 80° C. until reaching a final steadyvalue of approximately 25° C. (room temperature).

Those of skill in the art recognize that retention of molecules within apolymer matrix can be enhanced if the matrix is selectively permeable,i.e., the matrix allows diffusion of smaller molecules but not largerone. For example, in some embodiments, in order to prevent the passageof antibodies and other proteins having a molecular weight greater than30,000 Daltons (Da) through the matrix but allowing passage of nutrientsessential for cellular growth and metabolism, a useful permeability ofthe article is in the range of between 10,000 Da and 100,000 Da, forexample.

The speed of erosion of a scaffold produced from a bioerodible orbiodegradable polymer can be related to the molecular weights of thepolymer. Higher molecular weight polymers (e.g., with average molecularweights of 90,000 Da or higher) can produce scaffolds which retain theirstructural integrity for longer periods of time, while lower molecularweight polymers (e.g., average molecular weights of 30,000 Da or less)can produce scaffolds which can erode much more quickly.

In some embodiments, additional features, such as roughened spots,pores, holes, etc, can be introduced into the scaffolds by machiningmilling, grinding, etc. to promote osteoconductive growth. Cells canreadily migrate and attach upon such roughened surfaces. Introduction ofpores into the compositions of the invention may also be used toregulate permeability, degradation rate, and mechanical properties ofthe articles disclosed herein. For example, pores may be introducedmechanically or chemically into the polymer matrix. In some embodiments,pores are introduced mechanically, such as by machining (e.g., punching)holes in a film that is subsequently stacked or rolled as describedherein. In some embodiments, pores are introduced chemically byincorporating a porogen into the polymer and subsequently removing itonce the polymer matrix has formed. In some embodiments, the sublimationporogen is removed by reducing pressure, such as removal using a vacuum.Vacuum pressure can be less than 100 millibars, less than 50 millibars,less than 25 millibars, less than 20 millibars, less than 15 millibars,less than 10 millibars, less than 5 millibars, less than 1 millibar, orless. In some embodiments, the sublimation porogen is removed along withwater, e.g., drying a gel under a vacuum as discussed supra, and, thus,removing both water and the porogen at the drying temperatures andpressures disclosed herein. Notwithstanding the method of introduction,pores may be closed (i.e., pores not forming a contiguous space withother pores or the surface) or interconnected (i.e., pores form acontiguous space with other pores or the surface). In some embodiments,the compositions of the invention comprise interconnected pores.

Structural elastomeric and/or pliant films, grafts, and scaffolds fortissue regeneration applications are readily applicable to orthopedics,neurosurgery, and maxillofacial surgery, prosthetic tissue interface, aswell as other clinical disciplines. Other useful articles that can beprovided by the present invention include tissue engineered scaffoldsand grafts, packing materials, fixation devices, surgical guides,prosthetic/tissue interfaces, plates, screws, sutures, staples, barbsand clips. Disclosed herein are systems, compositions, and methodsuseful for making and using scaffolds, which may be implanted at adesired location and can be utilized as xenografts, allografts,artificial organs, or other cellular transplantation therapeutics. Thesescaffolds can be used to induce a desired configuration of cellattachment/tissue formation at a specified location. The scaffold may bea permanent or long-term implant or may degrade over time as the host'snatural cells replace the scaffold. The scaffold may be created in situ,or may be pre-fabricated and implanted into a patient, at a desiredlocation using minimally invasive techniques.

In some embodiments, the blood-derived plastic articles disclosed hereinmay be used to create bioresorbable wound dressings or band-aids. Wounddressings may be used as a wound-healing dressing, a tissue sealant(i.e., sealing a tissue or organ to prevent exposure to a fluid or gas,such as blood, urine, air, etc., from or into a tissue or organ), and/ora cell-growth scaffold. In some embodiments, the wound dressing mayprotect the injured tissue, maintain a moist environment, be waterpermeable, easy to apply, not require frequent changes, be non-toxic, benon-antigenic, maintain microbial control, and/or deliver effectivehealing agents to the wound site. Wound dressings may be used inconjunction with wound repair applications, for example orthopedicapplications, such as bone filling/fusion for osteoporosis and otherbone diseases; cartilage repair for arthritis and other joint diseases;tendon repair; for soft tissue repair, including nerve repair, organrepair, skin repair, vascular repair, muscle repair; and ophthalmicapplications. In some embodiments, wound dressings may be used inassociation with any medical condition that requires coating or sealingof a tissue. For example, lung tissue may be sealed against air leakageafter surgery; leakage of blood, serum, urine, cerebrospinal fluid, air,mucus, tears, bowel contents, or other bodily fluids may be stopped orminimized; barriers may be applied to prevent post-surgical adhesions,including those of the pelvis and abdomen, pericardium, spinal cord anddura, tendon, and tendon sheath, treating exposed skin, in the repair orhealing of incisions, abrasions, burns, inflammation, and otherconditions requiring application of a coating to the outer surfaces ofthe body, applying coatings to other body surfaces, such as the interioror exterior of hollow organs, including blood vessels, cardiovascularsurgery applications, thoracic surgery applications, neurosurgeryapplications, general surgery applications, repair in general trauma,plastic surgery applications, ophthalmic applications, orthopedicsurgery applications, gynecology/obstetrics applications, prevention ofadhesions, urology applications, dental surgery applications, and repairof incisions and other openings made for surgical purposes.

In some embodiments, the wound can be cultured to determine whetherinfection is present. The wound tissue can be debrided, if needed. Ifthe culture is positive, the wound can be treated for the infection, forexample by applying an antibiotic prior to or concurrently withapplication a blood plasma-derived plastic article of the presentinvention. Exemplary antibiotics include, but are not limited to,penicillin or cephalosporin. Where the culture is negative, noantibiotics need to be applied, and the wound is treated with the bloodplasma-derived plastic article of the invention. For example, powder ora sheet of a blood-derived plastic article can be applied to the woundin any of a variety of formulations disclosed herein, and the wound canbe dressed with conventional wound dressings, such as COMPEEL™ wounddressing, DUODERM™ wound dressing, TAGADERM™ wound dressings or OPSITE™wound dressing. Dressings can be changed at intervals ranging between 1day and 5 days, and may be changed at intervals of 3-4 days. Dependingon the extent of damage to the underlying tissue, healing of partialthickness defect wounds can be observed in as little as 4 days and offull thickness defect wounds in as little as 2-4 weeks.

In some embodiments, the present invention provides methods forpromoting healing of a skin wound comprising: applying to the skin woundsurface an effective amount of a blood-derived plastic article, whereinthe blood-derived plastic article comprises at least one biologicalresponse modifier. An effective amount of blood-derived plastic articlecan be that amount readily ascertainable by a skilled physiciansufficient to promote or facilitate healing of the skin wound.

In some embodiments, the present invention provides methods forpromoting healing of a tissue wound or defect comprising: applying tothe tissue wound or defect an effective amount of a blood-derivedplastic article, wherein the blood-derived plastic article comprises atleast one biological response modifier.

In some embodiments, the present invention provides methods forproviding a resorbable graft to a graft position in a subject,comprising: inserting a blood-derived plastic article into a graftposition in a subject, wherein the blood-derived plastic articlecomprises at least one biological response modifier.

In some embodiments, the present invention provides methods fordelivering stem cells to a tissue of a subject, comprising: contacting ablood-derived plastic article comprising stem cells with a tissue of asubject. The stem cells can be autologous, allogeneic, or xenogenic. Thestem cells can be embryonic and/or adult. The stem cells can be seededonto or within the blood-derived plastic article by dispersing the stemcells on top of the article or soaking the article in a compositioncomprising the stem cells. The article can be placed directly in contactwith the tissue or cultured for a period of time to increase theconcentration of stem cells therein.

In some embodiments, the present invention provides methods forconnecting a first portion of a tissue with a second portion of atissue, comprising: contacting at least one blood-derived plasticarticle selected from the group consisting of a suture, staple and barbwith a first portion of a tissue with a second portion of a tissue suchthat the first portion of the tissue and the second portion of thetissue are connected. In some embodiments, the blood-derived plasticarticle can form bonds with the tissue to provide temporary or prolongedconnection to the tissue. The tissues can be of the same or dissimilartypes, for example the article can be used to connect portions of skintissue or a portion of a bone tissue to a tendon tissue.

In some embodiments, the blood-derived plastic articles of the presentinvention may be used to fabricate coatings for devices to be used inthe body or in contact with bodily fluids, such as medical devices,surgical instruments, diagnostic instruments, drug delivery devices, andprosthetic implants. Coatings may be fabricated directly on such objectsor may be pre-fabricated in sheets, films, blocks, plugs, or otherstructures and applied/adhered to the device.

In some embodiments, the blood-derived plastic articles of the presentinvention may be placed into a seeping wound to seal off the blood flow.Such wound plug or blood clotting applications may be particularlyuseful, for example, in battlefield applications.

In some embodiments, the blood-derived plastic articles of the presentinvention may be fabricated to provide delivery of a therapeutic agent,such as a biological response modifier and/or drug, at a desiredlocation. Therapeutic agents may be included in a coating as anancillary to a medical treatment (for example, antibiotics) or as theprimary objective of a treatment (for example, a gene to be locallydelivered).

Examples of useful tissue-engineered constructs can include elastomericsheets such as layered, rolled or tube structures and machined sheetswhich may include holes, possibly of defined geometries or patterns, tofacilitate host tissue interstitial communication throughout theconstruct. Topical applications of sheet materials may include, withoutlimitation, skin substitutes following burn and chronic non-healingwounds/sores; surgical soft tissue defect fillers; post skin and breastcancer resection; plastic surgery related applications to help minimizescarring; and dental applications, including guided tissue regeneration.Interior (rather than topical) applications include duraplasty,peripheral nerve guides, adhesion prevention in various applicationssuch as gastrointestinal and cardiovascular surgery, hernia repair,degradable thermal insulators for cryosurgery, renal applications,anastomoses, tendon/ligament repair, heart valves and patches, bursarepair to prevent adhesions, and drug delivery of growth factors,analgesics, chemotherapeutics, antibiotics and other drugs via implantedreservoirs or impregnated plastics with or without pores.

Solid forms of the present materials (with solid ranging from rubberyplastic to very hard plastic) may be used for any of the above-mentionedapplications or also in fillers or shaped grafts for craniofacial,dental, orthopaedic, neurosurgical and plastic surgical applications; orin “granular” filler, tubes and other shapes to fill defects due totrauma, cancer resection, spinal fusion, cranial defect, diseased ordegraded joints such as due to arthritis or osteonecrosis; or inresorbable implants for arthroplasty, prosthetic-to-prostheticinterfaces; degradable screws, plates and other fixation devices;cartilage and meniscus graft applications; to provide fillers forcartilage defects; to create intervertebral disks to use as replacementsfor failed or failing disks; and to create bone resurfacing molds. Solidforms may also be used in tissue engineering applications, withcapability also to deliver cells and/or growth factors for a wide rangeof tissue types. Such autogenic blood-derived plastic scaffolds may alsobe formed so as to incorporate autogenic adult stem cells. With the everincreasing potential applications of stem cells, the structuresdescribed herein could meet the demand for scaffolds capable ofdelivering stem cells for other than hematopoeitic stem cellapplications. Microbarbs can be used for attaching graft materials,including corneal grafts, cartilage grafts, for blood vessel and othertubular structure anastomoses. Finally, for cell culture applicationsPBP wafers can be constructed and placed in cell culture dishes, orporous spheres can be suspended in cell culture.

Conventional bone grafts, including autografts, allografts andsynthetics are far from ideal, yet these are currently the second mostimplanted of all biomaterials (blood products are first). Autologous andallogeneic plastics could economically address many of the problemsassociated with the current options. Beyond bone grafts, there are manyother important applications, such as nerve guides, prosthetics/tissueinterfaces, tendon repair, and wound protection bandages. A potentialbusiness model is an integrated plastics manufacturing system forhospitals that can be placed in or adjacent existing blood banks orbatch manufacturing at any location, including (as recited above) acentrifuge, a dryer, a powder miller, disposable molds in standardshapes, compression molds and a cooperating hot press, and a vacuumdegasser, as discussed above.

Sterilization of PBPs can be performed throughout processing, rangingfrom screening of plasma based on established donor collectionprotocols, by techniques known and developing for bacterial and viralminimization, alcohol, gamma- or other sterilization techniques ofplasma powder and/or final post-packaging that represents minimal lossof biological activity, such as gamma radiation and ethylene oxide gas.

As discussed above, allogeneic grafts (such as bone grafts) have severallimitations, including high variability of graft quality from donor todonor. It would be desirable to have a means to perform qualityassessment (QA) and/or quality control (QC) of allogeneic graftmaterials with respect to the presence and/or amount of biologicalresponse modifier, such as growth factor(s), in each graft.

In some embodiments, the present invention provides methods that can beused for providing quality assessment (QA) and/or quality control (QC)of allogeneic articles, such as graft materials, with respect to thepresence and/or amount of biological response modifier(s) in eacharticle. These methods are not only applicable to assist in preparationof blood-derived plastic articles as described above, but also to anyarticle(s) comprising biological response modifier(s) which are preparedfrom allogeneic blood or blood component sources.

Generally, the methods involve determining a range of acceptableconcentrations of a selected biological response modifier for a batch ofblood, measuring the concentration of the selected biological responsemodifier in each batch of blood from which an article is to be prepared,and comparing the measured concentration to determine if the measuredconcentration falls within the range. If the measured concentration iswithin the range, then the batch is acceptable for use in a compositionto prepare an article. If the measured concentration is below the range,then the amount of biological response modifier in the batch can beadjusted by adding supplemental biological response modifier or mixingwith batch(es) of blood having higher concentration(s) of the biologicalresponse modifier, or electing not to use the batch. If the measuredconcentration is above the range, then the amount of biological responsemodifier in the batch can be adjusted by removing excess biologicalresponse modifier or mixing with batch(es) of blood having lowerconcentration(s) of the biological response modifier, or electing not touse the batch. Accordingly, batches can be selected to provide desiredconcentration(s) of the blood modifier.

Generally, to determine the range of acceptable concentrations of theselected biological response modifier for a batch of blood, theconcentration of the biological response modifier can be measured foreach of a plurality of blood batches; articles can be prepared from eachof the respective blood batches in a manner such as those describedabove; the concentration of the biological response modifier for each ofthe articles can be determined; a range of acceptable concentrations ofthe biological response modifier in an article can be determined; andthe range of acceptable concentrations of the biological responsemodifier in the article can be correlated to a respective range ofacceptable concentration of the biological response modifier in a bloodbatch.

Thus, in some embodiments, the present invention provides methods forassessing the concentration of a biological response modifier in anarticle comprising: (a) determining a range of acceptable concentrationsof a pre-determined biological response modifier for a batch of blood tobe used to prepare an article; (b) determining the concentration ofpre-determined biological response modifier in a blood batch to be usedto prepare an article; and (c) comparing the concentration determined in(b) to the range of acceptable concentrations obtained from (a). In someembodiments, the concentration of pre-determined biological responsemodifier can be adjusted in the blood batch that has a measured valuedetermined in step (b) which is above or below the range determined in(a) by adding more biological response modifier and/or blood frombatches having a higher or lower concentration of biological responsemodifier, as appropriate, to adjust the concentration accordingly. Thepresence and/or concentration of selected biological responsemodifier(s) in blood, a composition or article can be determined in amanner as discussed above, for example by corresponding assay.

In some embodiments, (a) above can be preceded by the following: (1)determining the concentration of the pre-determined biological responsemodifier for each of a plurality of blood batches; (2) determining theconcentration of the biological response modifier for each of aplurality of blood-derived articles prepared from each of the respectiveblood batches of (1); (3) determining an acceptable range ofconcentrations of the biological response modifier for the blood-derivedarticles based upon the concentrations determined in (2); and (4)correlating the acceptable range of concentrations of the biologicalresponse modifier for the blood-derived plastic articles obtained from(3) with the concentrations of the biological response modifier for theblood batches obtained in (1) to determine a range of acceptableconcentrations of the biological response modifier for the batch ofblood of (a).

In some embodiments, the age or other physical characteristics of thedonor(s) can be selected to provide blood having predetermined desiredcharacteristics corresponding to predetermined characteristics in theresulting articles. For example, donors of about 18 to about 30 years ofage can be selected to provide blood having predetermined levels of oneor more biological response modifiers.

The invention disclosed herein is exemplified by the followingpreparations and examples which should not be construed to limit thescope of the disclosure. Alternative mechanistic pathways and analogousstructures will be apparent to those skilled in the art.

EXAMPLES

In general, initial experiments were performed using rabbit and humanplasma testing such variables as dried plasma particle size, percentplasticizer (such as glycerol), plasma powder/plasticizer equilibrationtime, and processing temperature and pressure. Furthermore, ammoniumacetate porogen and genipen crosslinking validation experiments wereperformed. In general, as overall conclusions, when plasmapowder/plasticizer ratio is 55/45 and is held constant, and mixingequilibration time for dough mixing is varied, the resulting relativehardness of the bioplastic decreases as the dough incubation timeincreases. However, when plasticizer concentration is varied, whileholding dough mixing and processing temperature and pressure constant,such an approach results in a decrease in relative hardness of thebioplastic as the relative plasticizer concentration increases.

Example 1

As an example of initial biocompatibility of plasma-based plastics,plasma-based constituents (plasma powder/glycerol 55/45) were vibratomedto 300 micron thickness samples and sterilized via incubation in 70%ethanol for ten minutes. Human MG-63 human osteoblastic cells wereseeded upon samples and incubated for three days. Cell containingsamples were processed for scanning electron microscopy (SEM). Cellsexhibited ready binding, proliferation and migration upon the bioplasticsurface. Furthermore, cell proteolytic remodeling of the plastic wasreadily apparent and extensive cellular processes are interactingdirectly with the bioplastic, with proteolytic degradation creating aporous material from a smooth surface.

Example 2

Rabbit plasma bioplastic samples were prepared and placed in cell freeserum containing cell culture medium and held at 37° C. for up to 60days. Samples were weighed and measured for surface area at indicatedtimes. The bioplastic was found to swell about 50% upon addition tomedia but thereafter to remain constant in size throughout the durationof sampling. This indicates that the present bioplastic will notspontaneously degrade consistent with cell proteolytic degradation.

Example 3

Human plasma powder was sized into ≦38 micron and ≦150 microndistributions. Using similar processing conditions to those described inthe first sentence of Example 1, processed slurries werethermomechanically molded into micron peg molds. The smaller particlesize of ≦38 microns resulted in finer structural features compared toparticle sizes of ≦150. In some embodiments, useful particle size rangesfor the human plasma powder can be 38-500 microns, or 50-200 microns or75-150 microns.

Example 4 Retained Biological Activity in Plasma Based Plastics (PBPs)

In some embodiments, biological activity within PBPs can be retained byappropriate processing conditions. This biological activity can beprovided by growth factors and extracellular matrix (ECM) moleculescontributed by platelets and to a somewhat lesser extent the plasmaitself. An example of a processing parameter which can providebioplastics with substantially preserved biological activity ofbiological constituents is “low” temperature processing duringplastification. Such low temperature processing can be conducted at atemperature of less than about 65° C., or about 55° C. to about 65° C.,or about 60° C.

As shown in Table 1, as pressing time at 60° C. increases from 7.5 to 30minutes there was a significant loss in biological activity in theresulting PBPs. Biological activity was determined by taking knownquantities of PBP samples, pulverizing to powder under liquid nitrogen,extracting soluble growth factors from the powder, and determining theability of powder extracts to stimulate osteoblastic precursor cellproliferation in vitro.

TABLE 1 Effect of pressing time on biological activity of PBP PressingBiological Activity Time¹ (% above control)² Serum Control³  158⁴ PBP:7.5 min 285 PBP: 15 min 150 PBP: 30 min  48 ¹PBP pressed at 60 C. at10.7 kspi for indicated times ²% above non-serum, cell culture mediacontrol ³10% FBS in cell culture media ⁴Values represent the mean oftriplicate determinations

Example 5

The effect of pressure on biological activity of tested PBP samples isshown in Table 2. PBP samples subjected to the higher pressure of 14.7kspi had similar growth factor biological activity compared to samplessubjected to the lower pressure of 10.7 kspi.

TABLE 2 Effect of pressing pressure on biological activity of PBPPressing Biological Activity Pressure¹ (% above control)² Serum Control³ 148⁴ PBP: 60° C., 10.7 kspi  96 PBP: 60° C., 14.7 kspi 120 PBP: 65° C.,10.7 kspi 142 PBP: 55° C., 14.7 kspi 148 ¹PBP pressed at indicatedtemperature and pressure for 15 min ²% above non-serum, cell culturemedia control ³10% FBS in cell culture media ⁴Values represent the meanof triplicate determinations

Example 6

Another example of retained biological activity as well asbiocompatibility is depicted in FIG. 1. Osteoblastic precursor cellswere cultured on PBPs and then monitored for subsequent cellinteractions using scanning electron microscopy. Increasingmagnification depicted in FIG. 1A-1D illustrate positive cell-PBPinteraction with active remodeling of the PBP substrate.

Example 7 Genipin Modification of PBPs

As shown in this example, genipin can be added prior to plastification.Because transport of genipin is not an issue, crosslinking occurs duringplastification, stabilizing the PBPs and minimizing any swelling whenplaced in biological fluids. FIG. 2 demonstrates that dissolving genipincrystals in ethanol prior to addition to the bioplastic dough results ina more homogeneous distribution of crosslinking (the second line ofbioplastic samples is demonstrably more homogeneous than the top line).Note that when genipin is delivered in crystalline form, it firstdissolves locally within the forming PBP, resulting in “islands” thateventually create a non-homogenous distribution of crosslinking in PBPs.When genipin crystals or powder are solubilized in ethanol prior toadding to the bioplastic dough phase, a homogenous color change occursthroughout the PBPs creating a more monolithic product.

Within the context of delivering growth factors and other biologicalcomponents, although there is a slight loss in biological activity,substantial biological activity remains in genipin treated PBPs (Table3). Biological assessments were conducted as with Table 1 and 2. Thereis no difference between the forms of genipin added to the bioplasticdough, either crystalline or dissolved in ethanol.

TABLE 3 Effect of genipin on biological activity of PBP BiologicalActivity PBP Sample¹ (% above control)² Serum Control³   286 ± 22⁴ PBP:No Genipin   147 ± 2.5 PBP: 2% Genipin (powder) 105 ± 4 PBP: 2% Genipin(ETOH) 102 ± 5 PBP: ETOH 127 ± 6 ¹PBP pressed at 60 C., 10.7 kpsi for 15min ²% above non-serum, cell culture media control ³10% FBS in cellculture media ⁴Values represent the mean ± SEM of triplicatedeterminations

The inclusion of genipin in PBPs can have a significant influence on PBPmechanical properties. As shown in Table 4 below, the inclusion ofgenipin increased the Young's modulus of tested samples by 4-9 fold.

TABLE 4 Mechanical properties of PBPs Young's Modulus Max Stress %Genipin (MPa) (MPa) Powder 0 9 1.36 1 50 1.16 2 80 2.19 Powder + Water 09 0.8 1 40 1.2 2 60 1.1 Ethanol 2 40 2.4 PBP were 65/35 PRP/glycerol(w/w) pressed at 60° C., 10.7 kpsi for 15 min

Example 8 Lyophilized Plasma Particle Size on PBP Characteristics

As shown in FIG. 3, smaller particle size can provide more uniform moldfill. The top row of micrograph depictions of smaller PBP particle size(≦38 μm) show more uniform mold fill than larger PBP particle size (≦150μm). As the particle size becomes smaller this denotes a fasterequilibration time of “wetting” powder with added plasticizer during thedough preparation. These properties can be desirable during micromoldingor for micromachining preparation of PBP; whereas larger particle sizecan enable better macromolecular interlock between particles duringplastification, resulting in PBPs with acceptable mechanical properties.

Example 9 Addition of Calcium Phosphate Particulates to PBPs

Calcium phosphate particulates can be added during PBP dough preparationto create PBP with both organic and inorganic components. FIG. 4 showsthe addition of up to 10% nanoparticulate tricalcium phosphate (TCP)powder during dough formation with an increase in PBP opacity as TCPconcentration increases. Alternatively, other clinically relevant formsof calcium phosphate, including but not limited to hydroxyapatite, canbe substituted or mixed with TCP. The inclusion of such materials canalter mechanical properties, degradation, growth factor release rates,and provide additional osteoconductivity.

Example 10 Uncrosslinked PRP-PBP is Stable Under In Vitro Conditions

PRP based PBP was placed under simulated in vivo conditions, 37° C. inserum containing media for 60 days. A slight swelling occurred withinthe first day, but there was no subsequent change throughout theincubation period.

Example 11 Plasma Bioplastic Containing 65/35 PRP/Glycerol

Human plasma was clotted with calcium chloride by adding 1 part IMcalcium chloride in water to 52.6 parts human plasma. The clot was thenlyophilized (˜6 mTorr) for 72 hours to a water content of 8% by weight.Plasma powder was achieved by grinding the dried material in amechanical grinder then sieving through a 150 μm sieve. To formulate theplastic, 650 mg of plasma powder and 350 mg of glycerol were added to asmall beaker. The components were mixed until homogeneous and allowed toincubate at room temperature in a closed container for approximately 21hours. The resulting “dough” was pressed in a 13 mm diameter cylindricalpress at 59° C. and 2200 lbs of pressure (10.7 kpsi) for 10 minutes. Thebioplastic product (13 mm diameter×˜7 mm tall) was cut into 1 mm thickslices and then seeded with human MG-63 pre-osteoblast cells. Following4 days of growth, analysis by scanning electron microscopy (SEM)demonstrated cell proliferation and positive interaction with theplastic, as indicated by cell-mediated degradation of the bioplastic aswell as multiple cellular processes interacting with the bioplastic.

Example 12 Plasma Bioplastic Containing 10% TCP and 0.5% GenipinCrosslinker

585 mg of plasma powder (described in Example 1) and 100 mg ofbeta-tricalcium phosphate (TCP) were added to a small beaker andthoroughly mixed with a spatula. Glycerol (315 mg) was added, followedby 73.2 μL of 68.4 mg/mL genipin dissolved in ethanol. The componentswere mixed until homogeneous and allowed to incubate at room temperaturein a closed container for approximately 21 hours. The resulting “dough”was pressed in a 13 mm diameter cylindrical press at 59° C. and 2200 lbsof pressure (10.7 kpsi) for 10 minutes. The bioplastic product (13 mmdiameter×˜7 mm tall) was cut into 1 mm thick slices and then seeded withhuman MG-63 pre-osteoblast cells. Following one week of growth, theconstruct was analyzed by scanning electron microscopy (SEM) andtransmission electron microscopy (TEM). Cells were shown to completelycover the bioplastic, exhibiting multiple cell layers as well asbioplastic degradation and invasion into the bioplastic.

Example 13 Plasma Bioplastic with Ammonium Acetate (50% w/w) and 0.75%Genipin

425 mg of plasma powder (described in Example 1) and 500 mg of ammoniumacetate were added to a small beaker and thoroughly mixed with aspatula. Glycerol (75 mg) was added, followed by 54.9 μL of 136.7 mg/mLgenipin dissolved in ethanol. The components were mixed untilhomogeneous and allowed to incubate at room temperature in a closedcontainer for approximately 21 hours. The resulting “dough” was pressedin a 13 mm diameter cylindrical press at 59° C. and 2200 lbs of pressure(10.7 kpsi) for 10 minutes. The bioplastic product (13 mm diameter×˜7 mmtall) was sliced into 1 mm thick slices, which were placed in a vacuum(˜6 mTorr) for 48 hours to sublimate and remove the ammonium acetate.The resulting bioplastic was a porous material with 300-400 μm poresize.

Example 14 Composite Plasma Bioplastic with Differential Porosity

Plasma plastics can be made having regions comprised of differentchemical or physical properties and/or materials. In this example, threedoughs were prepared containing different porogen concentrations. Whenlayered prior to compression, the final treated product containeddifferent porosities across the sample. In dough #1, 500 mg of plasmapowder (described in Example 11) was first mixed with 500 mg dextroseand then 300 mg glycerol and 100 μL of 68.4 μg/mL genipin, dissolved inethanol. In dough #2, 750 mg of plasma powder (described in Example 11)was first mixed with 190 mg dextrose and then 280 mg glycerol and 94 μLof 68.4 μg/mL genipin dissolved in ethanol. In dough #3, 650 mg ofplasma powder (described in Example 11) was mixed only with 350 mgglycerol and 73.2 μL of 68.4 μg/mL genipin dissolved in ethanol. Eachrespective dough was, mixed until homogeneous and allowed to incubate atroom temperature in separate closed containers for approximately 21hours. The resulting doughs were stacked (first dough on top, thirddough in the middle, and second dough on the bottom) and pressed in a 13mm diameter cylindrical press at 59° C. and 2200 lbs of pressure (10.7kpsi) for 10 minutes. Slices of the resulting plastic were soaked in PBS(phosphate buffered saline) for two days followed by one day in ddH₂O(distilled deionized water), after which the samples were frozen at −20°C. and lyophilized overnight. The structure of each dried sample wasthen analyzed by scanning electron microscopy. The analysis showeddiscrete regions of porosity (boundary of dough vs. dough #3 materialand dough #3 vs. dough #2 material) as well as differential porosities(dough #3 vs. dough #2 material).

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications which are within the spirit and scopeof the invention, as defined by the appended claims.

What is claimed is:
 1. A blood-derived plastic article comprising atleast partially dried and clotted whole plasma, at least one biologicalresponse modifier, and at least one drug selected from the groupconsisting of an antibiotic, an analgesic, an anti-inflammatory, animmunosuppressive, an immunomodulator, and mixtures thereof.
 2. Theblood-derived plastic article according to claim 1, wherein the at leastone drug is an antibiotic.
 3. The blood-derived plastic articleaccording to claim 2, wherein the antibiotic is a heat sensitiveantibiotic.
 4. The blood-derived plastic article according to claim 2,wherein the antibiotic is selected from the group consisting ofcephalosporins, penicillins, sulfonamides, and tetracyclines.
 5. Theblood-derived plastic article according to claim 2, wherein theantibiotic is an aminoglycoside antibiotic.
 6. The blood-derived plasticarticle according to claim 5, wherein the aminoglycoside antibiotic isselected from the group consisting of gentamicin and neomycin.
 7. Theblood-derived plastic article according to claim 2, wherein theantibiotic is a glycopeptide antibiotic.
 8. The blood-derived plasticarticle according to claim 7, wherein the glycopeptide antibiotic isvancomycin.
 9. The blood-derived plastic article according to claim 2,wherein the antibiotic is a macrolide antibiotic.
 10. The blood-derivedplastic article according to claim 9, wherein the macrolide antibioticis selected from the group consisting of azithromycin andclarithromycin.
 11. The blood-derived plastic article according to claim2, wherein the antibiotic is a quinolone antibiotic.
 12. Theblood-derived plastic article according to claim 11, wherein thequinolone antibiotic is selected from the group consisting ofciprofloxacin, gatifloxacin, and levofloxacin.
 13. The blood-derivedplastic article according to claim 2, wherein the antibiotic is presentin the blood-derived plastic article in a concentration gradient. 14.The blood-derived plastic article according to claim 1, wherein the atleast one drug is a steroidal anti-inflammatory.
 15. The blood-derivedplastic article according to claim 1, wherein the blood-derived plasticarticle comprises at least two drugs, wherein at least one drug is anantibiotic and at least one drug is an analgesic.
 16. The blood-derivedplastic article according to claim 15, wherein the blood-derived plasticarticle comprises an antibiotic and an analgesic.
 17. The blood-derivedplastic article according to claim 15, wherein the antibiotic and theanalgesic are present in the blood-derived plastic article in aconcentration gradient.
 18. The blood-derived plastic article accordingto claim 1, wherein the blood-derived plastic article comprises at leastthree drugs, wherein at least one drug is an antibiotic, at least onedrug is an anti-inflammatory, and at least one drug is an analgesic. 19.The blood-derived plastic article according to claim 18, wherein theblood-derived plastic article comprises an antibiotic, ananti-inflammatory, and an analgesic.
 20. The blood-derived plasticarticle according to claim 18, wherein the anti-inflammatory is presentin the blood-derived plastic article in a concentration gradient. 21.The blood-derived plastic article according to claim 18, wherein theanalgesic is present in the blood-derived plastic article in aconcentration gradient.
 22. The blood-derived plastic article accordingto claim 18, wherein the antibiotic, anti-inflammatory, and analgesicare present in the blood-derived article in a concentration gradient.23. The blood-derived plastic article according to claim 1, wherein theat least one drug is lidocaine.
 24. The blood-derived plastic articleaccording to claim 1, comprising a concentration gradient of the atleast one drug.
 25. The blood-derived plastic article according to claim1, wherein the at least one biological response modifier is present inthe blood-derived article in a concentration gradient.
 26. Theblood-derived plastic article according to claim 1, wherein the at leastone biological response modifier and the at least one drug are presentin the blood-derived article in a concentration gradient.
 27. Theblood-derived plastic article according to claim 1, wherein the articleis in the form of a film, patch, powder, putty, or solid block.
 28. Theblood-derived plastic article according to claim 1, wherein the at leastone biological response modifier is a bioactive protein selected fromthe group consisting of hormones, growth factors, cytokines,extracellular matrix molecules, and mixtures thereof.
 29. Theblood-derived plastic article according to claim 28, wherein thebioactive protein comprises at least one growth factor selected from thegroup consisting of platelet derived growth factors (PDGF), acidic andbasic fibroblast growth factors, transformation growth factor beta(TGF-beta), insulin like growth factors (IGF), epidermal growth factors(EGF), platelet-derived angiogenesis factors (PDAF), platelet-derivedendothelial growth factors (PDEGF), tumor necrosis factor-alpha (TNF-α),tumor necrosis factor-beta (TNF-β), vascular endothelial growth factors(VEGF), epithelial cell growth factors (ECGF), granulocyte-colonystimulating factors (G-C SF), granulocyte-macrophage colony stimulatingfactors (GM-CSF), nerve growth factors (NGF), neurotrophins,erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF-8), growthdifferentiation factor-9 (GDF9), hepatocyte growth factors (HGF),platelet factors, and mixtures thereof.
 30. The blood-derived plasticarticle according to claim 28, wherein the bioactive protein comprisesat least one extracellular matrix molecule selected from the groupconsisting of osteocalcin, osteonectin, fibrinogen, vitronectin,fibronectin, thrombospondin 1 (TSP-1), bone sialoprotein (BSP),proteoglycans, and mixtures thereof.
 31. The blood-derived plasticarticle of claim 1, wherein the platelet concentration of the wholeplasma in the blood-derived plastic article is increased compared to abaseline platelet concentration of whole plasma.
 32. The blood-derivedplastic article according to claim 1, wherein the blood-derived plasticarticle further comprises at least one plasticizer.
 33. Theblood-derived plastic article according to claim 32, wherein the atleast one plasticizer is selected from water and glycerol.
 34. Theblood-derived plastic article according to claim 33, wherein the atleast one plasticizer is glycerol.
 35. The blood-derived plastic articleaccording to claim 32, wherein the at least one plasticizer is presentin an amount of about 0.1 to about 80 percent by weight of theblood-derived plastic article.
 36. The blood-derived plastic articleaccording to claim 1, wherein the blood-derived plastic article furthercomprises at least one crosslinking agent.
 37. The blood-derived plasticarticle according to claim 36, wherein the at least one crosslinkingagent is genipin.
 38. The blood-derived plastic article according toclaim 37, wherein the at least one crosslinking agent is present in anamount of about 0.01 to about 20 percent by weight of the blood-derivedplastic article.
 39. A blood-derived plastic article comprising at leastpartially dried and clotted whole plasma, at least one biologicalresponse modifier, and at least one antibiotic.
 40. The blood-derivedplastic article of claim 39, wherein the at least one antibiotic isselected from the group consisting of an aminoglycoside antibiotic and aglycopeptide antibiotic.
 41. The blood-derived plastic article accordingto claim 40, wherein the aminoglycoside antibiotic is selected from thegroup consisting of gentamicin and neomycin.
 42. The blood-derivedplastic article according to claim 40, wherein the glycopeptideantibiotic is vancomycin.
 43. The blood-derived plastic articleaccording to claim 39, wherein the at least one biological responsemodifier is a bioactive protein selected from the group consisting ofhormones, growth factors, cytokines, extracellular matrix molecules, andmixtures thereof.
 44. The blood-derived plastic article according toclaim 43, wherein the bioactive protein comprises at least one growthfactor selected from the group consisting of platelet derived growthfactors (PDGF), acidic and basic fibroblast growth factors,transformation growth factor beta (TGF-beta), insulin like growthfactors (IGF), epidermal growth factors (EGF), platelet-derivedangiogenesis factors (PDAF), platelet-derived endothelial growth factors(PDEGF), tumor necrosis factor-alpha (TNF-α), tumor necrosis factor-beta(TNF-β), vascular endothelial growth factors (VEGF), epithelial cellgrowth factors (ECGF), granulocyte-colony stimulating factors (G-CSF),granulocyte-macrophage colony stimulating factors (GM-CSF), nerve growthfactors (NGF), neurotrophins, erythropoietin (EPO), thrombopoietin(TPO), myostatin (GDF-8), growth differentiation factor-9 (GDF9),hepatocyte growth factors (HGF), platelet factors, and mixtures thereof.45. The blood-derived plastic article according to claim 43, wherein thebioactive protein comprises at least one extracellular matrix moleculeselected from the group consisting of osteocalcin, osteonectin,fibrinogen, vitronectin, fibronectin, thrombospondin 1 (TSP-1), bonesialoprotein (BSP), proteoglycans, and mixtures thereof.
 46. Theblood-derived plastic article of claim 39, wherein the plateletconcentration of the whole plasma in the blood-derived plastic articleis increased compared to a baseline platelet concentration of wholeplasma.
 47. The blood-derived plastic article according to claim 39,wherein the blood-derived plastic article further comprises at least oneplasticizer.
 48. The blood-derived plastic article according to claim47, wherein the at least one plasticizer is selected from water andglycerol.
 49. The blood-derived plastic article according to claim 48,wherein the at least one plasticizer is glycerol.
 50. The blood-derivedplastic article according to claim 47, wherein the at least oneplasticizer is present in an amount of about 0.1 to about 80 percent byweight of the blood-derived plastic article.
 51. The blood-derivedplastic article according to claim 39, wherein the blood-derived plasticarticle further comprises at least one crosslinking agent.
 52. Theblood-derived plastic article according to claim 51, wherein the atleast one crosslinking agent is genipin.
 53. The blood-derived plasticarticle according to claim 51, wherein the at least one crosslinkingagent is present in an amount of about 0.01 to about 20 percent byweight of the blood-derived plastic article.